Compositions and methods for prevention and treatment of hearing loss

ABSTRACT

Methods and compositions using an inhibitor of EGFR signaling for prevention or an inhibitor of EGFR signaling and a nucleic acid molecule encoding an atonal-associated factor for treatment of hearing loss are described.

INTRODUCTION

The patent application is a U.S. National Stage application of PCTApplication No. PCT/US2018/030114, filed Apr. 30, 2018, which claims thebenefit of priority from U.S. Provisional Patent Application Serial No.62/500,667, filed May 3, 2017, the contents of each of which are herebyincorporated by reference in their entirety.

This invention was made with government support under Grant NumbersDC006471 DC015010, DC015444, DC013879, DC013232, and CA021765 awarded bythe National Institutes of Health and Grant Numbers N00014-09-V-1014,N00014-12-V-0191, N00014-12-V-0775, and N00014-16-V-2315 awarded by theOffice of Naval Research. The government has certain rights in theinvention.

BACKGROUND

The ear is a complex organ composed of a labyrinth of structuresresponsible for hearing and balance. Perception of both hearing andbalance lies in the ability of inner ear structures to transformmechanical stimuli to impulses recognized by the brain. The sensoryreceptors responsible for hearing are located in the cochlea, aspiral-shaped canal filled with fluid. Within the cochlea is the organof Corti, which is lined with columnar sensory hair cells bridging thebasilar membrane and the tectorial membrane. As sound waves pass throughthe organ of Corti, the basilar membrane vibrates causing the hair cellsto bend back and forth. The movement depolarizes the hair cell, leadingto release of neurotransmitters to the auditory nerve, which carries theimpulse to the brain.

The inner-ear cochlear sensory epithelium is post-mitotic after birthand, in mice, exhibits only limited spontaneous regeneration during thefirst week after birth. Atonal BHLH Transcription Factor 1 (Atoh1), alineage-specific transcription factor for sensory hair cells, directlyconverts non-sensory supporting cells to sensory hair cells in cochlearexplant culture and in vivo (Gubbels, et al. (2008) Nature455(7212):537-41; Kelly, et al. (2012) J. Neurosci. 32(19):6699-710;Liu, et al. (2012) J. Neurosci. 32(19):6600-10; Liu, et al. (2014) PLoSOne 9(2):e89377; Zheng & Gao (2000) Nature Neurosci. (6):580-6).Further, the FDA has approved a clinical trial (NCT02132130) forassessing safety, tolerability and efficacy of CGF166, a recombinantadenovirus 5 (Ad5) vector containing a cDNA encoding the human Atoh1.However, it is not clear whether Atoh1-mediated non-sensory supportingcell-to-sensory hair cell conversion in vivo is efficient and completeand whether such conversion bypasses the progenitor-cell state orfollows normal developmental lineage paths precisely. In several mousemodels, Atoh1-converted sensory hair cells exhibited immature hair cellmorphology and did not express several terminal differentiation markers(e.g., Slc26a5 encoding prestin and Ocm encoding oncomodulin), and theconversion rate was low (6%-20%) (Kelly, et al. (2012) J. Neurosci.32(19):6699-710; Liu, et al. (2012) J. Neurosci. 32(19):6600-10; Liu, etal. (2014) PLoS One 9(2):e89377). In addition, in a subset of supportingcells marked by p75, epidermal growth factor receptor (EGFR) signalinghas been shown to be required for proliferation and down-regulation ofthe cell cycle inhibitor p27Kip1 (CDKN1b) to enable cell cycle re-entry(White, et al. (2012) Dev. Biol. 363:191-200).

SUMMARY OF THE INVENTION

The invention provides a method for the treatment or prevention ofhearing loss by administering to an animal in need thereof an inhibitorof epidermal growth factor receptor (EGFR) signaling. In accordance withtreatment, the method can further include administering an expressionvector harboring a nucleic acid molecule encoding an atonal-associatedfactor and/or other regenerative agents. In other embodiments, themethod further includes administering one or more otoprotective orregenerative agents. In further embodiments, the inhibitor of EGFRsignaling inhibits the expression or activity of EGFR, Ras, Raf, MEK,ERK/MAPK, JAK, STAT, PI3K, AKT, mTOR, NCK, PAK, JNK, PLC, PKC or a cellcycle-associated protein kinase inhibitor (e.g., Her-2, Aurora Kinase,B-Raf or PDGFR). In other embodiments, the inhibitor is an inhibitoryRNA, antibody or small organic molecule. Pharmaceutical compositions andkits containing an expression vector harboring a nucleic acid moleculeencoding an atonal-associated factor in combination with an inhibitor ofepidermal growth factor receptor (EGFR) signaling and optionally one ormore regenerative agents are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show that pharmacological inhibition of the EGFR signalingincreased Atoh1-induced hairy cell (HC) conversion in neonatal mousecochlear explants. Representative images show the immunostaining ofcochlear explants transfected with Atoh1-IRES-GFP and treated withvehicle (FIGS. 1A and 1C) or 100 nM AG1478 (FIGS. 1B and 1D). Expressionof hair cell marker Myo6 (↑) and Atoh1-transfected cells (GFP,

) are shown. FIGS. 1C and 1D represent high magnifications of the squareareas in FIGS. 1A and 1B, respectively. Scale bar: 100 μm in FIGS. 1Aand 1B, 20 μm in FIGS. 1C and 1D.

FIG. 2 shows quantification of Atoh1-induced HC conversion rate (Myo6+;GFP+/GFP+ cells in percentage) under different treatments indicated(n=1-5 for each treatment). Atoh1 is transfected (O/E) in allconditions.

FIG. 3 shows that the EGFR inhibitor MUBRITINIB (whose structure isshown) protects against cisplatin-induced hair cell loss in mousecochlear explants with IC₅₀ of 2.5 nM and LD₅₀ of >500 nM (TherapeuticIndex of >200). Number of explants: 1-4 at each dose; FVB cochlearexplants with 150 μM cisplatin treatment and middle turns were analyzed;curve fitting with R² of 0.86. Note that IC₅₀ values of MUBRITINIB wereconsistent in all assays demonstrating its specificity and potency.

FIG. 4 shows that the EGFR inhibitor Pelitinib (whose structure isshown) protects against cisplatin-induced hair cell loss. Pelitinib isan irreversible inhibitor of EGFR that exhibits protective effectsagainst cisplatin-induced Caspase-3/7 activity in HEI-OC1 cells withIC₅₀ of 0.6 μM (cisplatin-Glo 3/7) and LD₅₀ of >40 μM (CELLTITER-GLO).

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that inhibitors of EGFR and proteins downstream orassociated therewith protect hair cells damaged by cisplatin,antibiotics, noise, aging or other ototoxic insults and cansignificantly induce hair cell formation in conjunction with Atoh1overexpression in cochlear explant culture. Accordingly, this inventiveprovides compositions and methods for the prevention of hearing lossusing an inhibitor of EGFR and/or treatment of hearing loss using aninhibitor of EGFR in combination with Atonal-associated factor genetherapy. Ideally, the inventive methods prophylactically ortherapeutically treat an animal, preferably a mammal (e.g., a human),for at least one disorder associated with loss or damage of sensory haircells, e.g., disorders of the ear associated with damage of sensory haircells (such as hearing loss or balance disorders). The inventive methodsalso are useful in maintaining a level of sensory perception, i.e.,controlling the loss of perception of environmental stimuli caused by,for instance, the aging process or ototoxic agents.

EGFR Signaling.

Epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is acell-surface receptor activated by binding of its specific ligands,including epidermal growth factor (EGF), transforming growth factor(TGFα), HB-EGF, amphiregulin, betacellulin, epigen, and epiregulin. EGFRis a member of the ErbB family of receptors, a subfamily of four closelyrelated receptor tyrosine kinases: EGFR, HER2/c-neu (ErbB-2), Her3(ErbB-3) and Her4 (ErbB-4). Upon activation by its growth factorligands, EGFR undergoes a transition from an inactive monomeric form toan active homodimer. In addition to forming homodimers after ligandbinding, EGFR can pair with another member of the ErbB receptor family,such as ErbB2/Her2/neu, to create an activated heterodimer. EGFRdimerization stimulates its intrinsic intracellular protein-tyrosinekinase activity. As a result, autophosphorylation of several tyrosine(Y) residues in the C-terminal domain of EGFR occurs. These includeY992, Y1045, Y1068, Y1148 and Y1173. This autophosphorylation elicitsdownstream activation, signaling and/or expression ofRas/Raf/MEK/ERK/MAPK, JAK/STAT, PI3K/AKT/mTOR, NCK-PAK-JNK, PLC-DAG-PKCand/or a number of cell cycle-associated protein kinaseproteins/pathways. These signaling events initiate several signaltransduction cascades leading to DNA synthesis and cell migration,adhesion, and proliferation. Accordingly, “EGFR signaling” or “an EGFRsignaling pathway” refers herein to signaling by EGFR itself, as well asthe Ras/Raf/MEK/ERK/MAPK, JAK/STAT, PI3K/AKT/mTOR, NCK-PAK-JNK,PLC-DAG-PKC, cell cycle-associated protein kinase pathways/proteinsdownstream thereof.

Ras/Raf/MEK/ERK/MAPK Pathway.

The Ras/Raf/MEK/ERK/MAPK pathway (also known as the MAPK/ERK pathway) iswell-known in the art and plays a central role in regulating mammaliancell growth by relaying extracellular signals from ligand-bound cellsurface tyrosine kinase receptors such as EGFR. Activation of theMAPK/ERK (mitogen-activated protein kinase/extracellularsignal-regulated kinase) pathway is via a cascade of phosphorylationevents that begins with activation of Ras, e.g., HRas (GENBANK AccessionNo. NP_001123914, NP_001304983, or NP_789765), KRas (GENBANK AccessionNo. NP_004976 or NP_203524) or NRas (GENBANK Accession No. NP_002515).Activation of Ras leads to the recruitment and activation of Raf, e.g.,c-Raf or Raf-1 (GENBANK Accession No. NP_002871), A-Raf (GENBANKAccession No. NP_001243125, NP_001645 or NP_001243126) or B-Raf (GENBANKAccession No. NP_004324). Activated Raf then phosphorylates andactivates MEK1/2 (i.e., MAPK/ERK Kinase-1 and -2; GENBANK Accession Nos.NP_002746 and NP_109587, respectively), which then phosphorylates andactivates ERK1/2 (i.e., MAPK3/MAPK1; UniProt Accession Nos. P28482 andP27361, respectively). This chain of proteins, from Ras to ERK,communicates signals from cell-surface receptors to the DNA. ERKgenerates extensive changes in gene expression mediated by transcriptionfactors that control cell cycle progression, differentiation, proteinsynthesis, metabolism, cell survival, cell migration, and invasion andsenescence.

JAK/STAT Pathway.

The Janus kinase/signal transducers and activators of transcription(JAK/STAT) pathway stimulates cell proliferation, differentiation, cellmigration and apoptosis. Mechanistically, JAK/STAT signaling is composedof a few principal components. In mammals, the JAK family includes fourmembers: JAK1 (GENBANK Accession No. NP_001307852), JAK2 (GENBANKAccession No. NP_001309123 or NP_001309127), JAK3 (GENBANK Accession No.NP_000206) and Tyk2 (GENBANK Accession No. NP_003322). JAK activationoccurs upon ligand-mediated receptor multimerization thereby allowingtrans-phosphorylation. The activated JAKs subsequently phosphorylateadditional targets, in particular STATs. STATs are latent transcriptionfactors that reside in the cytoplasm until activated. The mammalianSTATs (i.e., STAT1, GENBANK Accession No. NP_009330 or NP_644671; STAT2,GENBANK Accession No. NP_005410 or NP_938146; STAT3, GENBANK AccessionNo. NP_003141, NP_644805, or NP_998827; STAT4, GENBANK Accession No.NP_001230764 or NP_003142; STAT5A, GENBANK Accession No. NP_001275647,NP_001275648, or NP_001275649; STAT5B, GENBANK Accession No. NP_036580;and STAT6, GENBANK Accession No. NP_001171549, NP_001171550,NP_001171551 or NP_001171552) bear a conserved tyrosine residue near theC-terminus that is phosphorylated by JAKs. This phosphotyrosine permitsthe dimerization of STATs through interaction with a conserved SH2domain. Phosphorylated STATs enter the nucleus and bind specificregulatory sequences to activate or repress transcription of targetgenes. Thus, the JAK/STAT cascade provides a direct mechanism totranslate an extracellular signal into a transcriptional response.

PI3K/AKT/mTOR Pathway.

The PI3K/AKT/mTOR pathway is an intracellular signaling pathwayimportant in regulating the cell cycle. Ligand-bound activation of EGFRleads to the activation of PI3K (phosphatidylinositol-4,5-bisphosphate3-kinase, e.g., Class 1 enzymes such as PIK3CA, PIK3CB, PIK3CG, PIK3CD,PIK3R1, PIK3R2, PIK3R3, PIK3R4, PIK3R5 and PIK3R6; Class 2 enzymes suchas PIK3C2A, PIK3C2B and PIK3C2G; and Class 3 enzyme PIK3C3). PI3Ksubsequently phosphorylates Akt (i.e., Protein Kinase B or PKB includingAKT1, UniProt Accession No. P31749; AKT2, UniProt Accession No. P31751;and AKT3, UniProt Accession No. Q9Y243). PIK3 subsequently activatesmTOR complexes, mTORC1 and mTORC2, which are each involved in cellgrowth. mTORC1, which is composed of mTOR, Raptor, GβL (mammalian lethalwith SEC13 protein 8) and domain-containing mTOR-interacting protein(DEPTOR), unifies multiple signals that indicate the availability ofgrowth factors, nutrients and energy in order to promote cellular growthand catabolic processes during stress. Active mTORC1 exerts numerousdownstream biological effects, including the translation of mRNA byphosphorylating downstream targets, such as 4E-BP1 and p70 S6 kinase,the suppression of autophagy through Atg13 and ULK1, ribosomebiogenesis, and activation of transcription that leads to increasedmitochondrial activity or adipogenesis. mTORC2, which is composed ofmTOR, Rictor, GβL, Sin1, PRR5/Protor-1 and DEPTOR, promotes cellsurvival through the activation of Akt. mTORC2 regulates cytoskeletaldynamics, ion transport and growth by activating PKCα andphosphorylating SGK1.

NCK-PAK-JNK Pathway.

Nck (non-catalytic region of tyrosine kinase adaptor protein 1; GENBANKAccession No. NP_001177725 or NP_001278928) is known to bind toactivated EGFR through its SH2 domain. Nck associates with PAK1(p21/CDC42/Rac1-Activated Kinase-1; GENBANK Accession No. NP_001122092or NP_002567) through the first N-terminal polyproline domain of PAK1and an SH3 domain of Nck. Nck activates PAK, which subsequentlyactivates JNKs (c-Jun Kinases) via MEKK1 (MAP/ERK Kinase Kinase-1;GENBANK Accession No. NP_005912) and MKK4/7 (MAP Kinase Kinase-4/7;GENBANK Accession No. NP_1268364, NP_003001, NP_001284484, orNP_001284485). Activated JNKs enter the nucleus and causephosphorylation of transcription factors such as c-Fos and c-Jun.

PLC-DAG-PKC Pathway.

Phospholipase C (PLC) ties EGFR activation to the generation ofsecondary messengers and calcium metabolism. EGFR recruits andphosphorylates PLC-γ1 (GENBANK Accession No. NP_002651 or NP_877963),which then generates diacylglycerol (DAG) andinositol-1,4,5-trisphosphate (IP3) from PtdIns(4,5)P2. DAG activatesmany isoforms of Protein kinase C (PKC), including conventional isoformsα, β, and γ, as well as PKC-ε and PKC-θ. PKC-α, PKC-β, PKC-γ, and PKC-εphosphorylate and activate c-Raf-1, thereby amplifying HRas/MEK1 andMEK2/ERK1/2 kinase cascades. PKC-θ activates Nuclear factor NF-kappa-Binhibitor kinase beta (IKK-beta) resulting in activation of the Nuclearfactor NF-kappa-B (NF-kB).

Cell Cycle-Associated Protein Kinases.

Protein kinases downstream or interacting with EGFR play a central rolein the regulation of the eukaryotic cell cycle. More specifically, theseprotein kinases are involved in signal transduction, chromosomecondensation, centrosome maturation, spindle assembly, spindleorientation, meiotic maturation, and cytokinesis. Accordingly, a“cell-cycle associated protein kinase” refers to a kinase downstream of,or interacting with, EGFR that regulates one or more of cell cycleprogression, cell division, cell proliferation, and cell cyclemachinery. In certain embodiments, the cell cycle-associated proteinkinase of this invention is Her2/neu, Aurora kinase, B-raf (as discussedherein) or platelet-derived growth factor receptor (PDGFR).

Her2/neu Kinase. Her2/neu is a 185-kDa transmembrane protein (GENBANKAccession No. NP_001005862, NP_001276865, NP_001276866, NP_001276867, orNP_004439) encoded by the erbB2 oncogene located on chromosome 17q21-22.Normal expression of Her2/neu at the cell surface is essential forregulating cell growth and epithelial cell survival. While a naturalligand of Her2/neu has not been identified, Her2/neu is known to be apreferred dimerization partner forming potent heterodimers with EGFR andHer3 (Lenferink, et al. (1998) EMBO J. 17:3385-97).

Aurora Kinase.

The Aurora kinases are a family of highly conserved serine/threoninekinases that are important for faithful transition through mitosis(Bischoff, et al. (1998) EMBO J. 17:3052-65; Carmena & Earnshaw (2003)Nat. Rev. Mol. Cell Biol. 4:842-54; Giet & Prigent (1999) J. Cell Sci.112:3591-601). The gene for Aurora A, maps to chromosome region 20q13.2,a region that has been found amplified in different human cancers.Aurora A (GENBANK Accession No. NP_001310232, NP_001310233,NP_001310234, NP_003591 or NP_940835) plays an important role incentrosome maturation, spindle assembly, meiotic maturation, andmetaphase I spindle orientation (Carmena & Earnshaw (2003) Nat. Rev.Mol. Cell Biol. 4:842-54). Aurora A function is regulated bydegradation, phosphorylation, and dephosphorylation, with its kinaseactivity dependent upon phosphorylation of threonine 288 (Thr288) in theactivation loop. Selective inhibition of Aurora A results in inhibitionof autophosphorylation of Aurora A at Thr288, monopolar spindles, andG2-M arrest (Girdler, et al. (2006) J. Cell Sci. 119:3664-75; Carpinelli& Moll (2008) Expert Opin. Ther. Targets 12:69-80). The Aurora B(GENBANK Accession No. NP_001243763, NP_001271455, NP_001300879,NP_001300880, or NP_001300881) gene maps to chromosome region 17p13.1and this kinase forms part of the chromosomal passenger complex (CPC)with three non-enzymatic subunits: inner centromere protein (INCENP),Survivin, and Borealin (Vader, et al. (2006) J. Cell Biol. 173:833-7).The highly dynamic CPC is critical for chromosome condensation,chromosome orientation on the mitotic spindle, through correctingchromosome-microtubule attachment errors, and the spindle-assemblycheckpoint (SAC), as well as the final stages of cytokinesis (Sampath,et al. (2004) Cell 118:187-20; Terada, et al. (1998) EMBO J. 17:667-76;Carmena, et al. (2012) Nat. Rev. Mol. Cell Biol. 13:789-803; Tanenbaum,et al. (2011) Curr. Biol. 21:1356-6). Aurora C (GENBANK Accession No.NP_001015878, NP_001015879, or NP_003151) expression has been reportedin testis, thyroid, and placenta and in meiotically dividing gametes(Ulisse, et al. (2006) Int. J. Cancer 119:275-82; Bernard, et al. (1998)Genomics 53:406-9; Kimura, et al. (1999) J. Biol. Chem. 274:7334-40;Yang, et al. (2010) Mol. Biol. Cell 21:2371-83). Further, nuclear EGFR,associated with STAT5, has been shown to bind and increase Aurora-A geneexpression (Hung, et al. (2008) Nucl. Acids Res. 36(13):4337-51).Overexpression of Aurora C has been suggested to induce abnormal celldivision resulting in centrosome amplification and multinucleation incells.

PDGFR Kinase.

PDGFR is involved in the control of cell proliferation, differentiationand survival in various tissues of vertebrates. Activated PDGFRphosphorylates itself and other proteins, and thereby engagesintracellular signaling pathways that trigger cellular responses such asmigration and proliferation. PDGFRα (UniProtKB Accession No. P16234) andPDGFRβ (GENBANK Accession No. NP_002600) are highly expressed in therapidly growing otocyst on embryonic days 12-14 and weakly expressedthereafter (Lee, et al. (2004) Acta Oto-Laryng. 124:558-62). Based uponthis analysis, it was suggested that integrity of PDGF signaling isrequired for the proliferation of developing cochlear hair cells (Lee,et al. (2004) Acta Oto-Laryng. 124:558-62). In addition, previousstudies have suggested that PDGF signaling is required for the trophismof the vascular and mesenchymal compartment in the neonatal mouse innerear and, indirectly, for the survival of the sensory epithelium(Hayashi, et al. (2008) Hear. Res. 245:73-81). Notably,heterodimerization and cross talk between the PDGFRβ and the EGFR hasindicated a role for EGFR transactivation in PDGF-stimulated cellmigration (Saito, et al. (2001) Mol. Cell Biol. 21(19):6387-94).

Inhibitors of EGFR Signaling.

An inhibitor of EGFR signaling is intended to refer to any molecule thatreduces, blocks or decreases the expression or activity of an EGFRprotein or a protein that interacts with or is in a downstream pathwayof EGFR, e.g., a Ras, Raf, MEK, ERK/MAPK, JAK, STAT, PI3K, AKT, mTOR(including a protein of an mTOR complex), NCK, PAK, JNK, PLC, PKC orcell cycle-associated protein kinase (e.g., Her-2, Aurora kinase, B-Raf,or PDGFR). An inhibitor of EGFR signaling also includes an inhibitorthat blocks the expression or activity of an EGFR ligand such as EGF,TGF-α, HB-EGF, AR, BTC, EPR, or epigen. In certain embodiments, theinhibitor of EGFR signaling inhibits or reduces the expression oractivity of EGFR, PLC, STAT3, JAK2, PI3K, MEK, Her-2, Aurora kinase,B-Raf, or PDGFR.

An inhibitor of this invention can selectively decrease or block theexpression of an EGFR signaling protein (i.e., transcription ortranslation of the protein), decrease or block the activity of an EGFRsignaling protein (i.e., binding to ligands, tyrosine kinase activity,phosphorylation, protein-protein interactions, and/or downstreamsignaling), decrease or block the biological effect(s) of an EGFRsignaling protein, and/or modify half-life or subcellular localization(membrane versus cytoplasmic or nuclear localization, internalization,and recycling) of an EGFR signaling protein. In particular, an inhibitorof EGFR signaling is an active agent that selectively decreases orblocks one or more of the following: transcription or translation,ligand binding, phosphorylation, multimerization, tyrosine kinaseactivity, internalization, and/or translocation into the nucleus.

Ideally, EGFR signaling is completely blocked, or is reduced by at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 92.5%, at least 95%, at least 97%, at least 98%, at least98.5%, at least 99%, at least 99.25%, at least 99.5%, or at least 99.75%by the inhibitor of EGFR signaling inhibitor as compared to normalphysiologic levels.

The inhibitor of EGFR signaling of this invention typically has a halfmaximal (50%) inhibitory concentration (IC₅₀) in the range of 1 pM to100 μM. Preferably, the inhibitor of EGFR signaling has an IC₅₀ value ofless than 10 μM, less than 5 μM, less than 1 μM, or less than 100 nM.Moreover, in some embodiments, the inhibitor of EGFR signaling isspecific/selective for one or more of the EGFR signaling proteins ofinterest and fails to inhibit, or inhibits to a substantially lesserdegree other non-EGFR pathway proteins. In this respect, it ispreferable that the inhibitor of EGFR signaling is a selective inhibitorof EGFR signaling. Preferably, selectivity is for one, two, three orfour EGFR signaling proteins and fails to inhibit, or inhibits to asubstantially lesser degree other non-EGFR pathway proteins. By way ofillustration, an inhibitor can be a dual EGFR and ERBB2 inhibitor, bothof which are EGFR signaling proteins. Methods for assessing theselectively of inhibitors are known in the art and can be based upon anyconventional assay including, but not limited to the determination ofthe IC₅₀, the binding affinity of the inhibitor (i.e., K_(i)), and/orthe half maximal effective concentration (EC₅₀) of the inhibitor forEGFR signaling protein of interest as compared to another protein(comparative protein). In particular embodiments, a selective inhibitorof EGFR signaling is an inhibitor that has an IC₅₀ value for an EGFRsignaling protein of interest that is at least twice or, more desirably,at least three, four, five, or six times lower than the correspondingIC₅₀ value for a comparative protein. Most desirably, a selectiveinhibitor of EGFR signaling has an IC₅₀ value for an EGFR signalingprotein which is at least one order of magnitude or at least two ordersof magnitude lower than the IC₅₀ value for a comparative protein.

An inhibitor of this invention can be a nucleic acid-based inhibitorsuch as an inhibitory RNA molecule (e.g., antisense molecule, aribozyme, siRNA, shRNA, miRNA, etc.); a protein that affects splicing or3′ processing (e.g., polyadenylation), or the level of expression ofanother gene within the cell (i.e., where gene expression is broadlyconsidered to include all steps from initiation of transcription throughproduction of a process protein), such as by mediating an altered rateof mRNA accumulation or transport or an alteration inpost-transcriptional regulation; an antibody (including fragments ormimetics); a peptide; a small organic molecule; or a combinationthereof.

The term siRNA refers to double stranded RNA or RNA and DNA species thatare active to reduce expression of targeted gene. These molecules areknown variously as “small interfering RNA,” “short interfering RNA” or“silencing RNA.” siRNA strands are usually 20-25 nucleotides long,although larger precursor molecules which are subject to cleavage invivo to form the active species are within the scope of the term as usedherein.

As used herein, an “miRNA molecule” or “miRNA” is a small RNA molecule,typically about 20 to 25 nucleotides, encoded by the genome of an animalor produced synthetically with a sequence which corresponds to oneencoded by the genome of the animal. As used herein, miRNA molecules maybe single-stranded or double-stranded.

When the inhibitor is, e.g., an inhibitory RNA, peptide or protein,nucleic acid molecules encoding such an inhibitor can be carried by thesame nucleic acid molecule that encodes the atonal-associated factor orcan be a separate nucleic acid molecule present on the same expressionvector or part of a different expression vector. Inhibitory RNAmolecules can be readily prepared based upon the nucleic acid sequencesdisclosed herein. Alternatively, inhibitory RNA molecules such as siRNAscan be obtained from commercial sources such as Dharmacon (see, e.g.,ON-TARGET plus siRNA SMART pools), Invitrogen or Zyagen. A decrease inthe expression of a protein can be measured using conventionaltechniques such as dot blot, northern blot, ELISA or western blotanalysis.

EGFR Inhibitors.

EGFR inhibitors that selectively decrease or block the expression ofEGFR itself include, but are not limited to, EGFR antisense, siRNA andmiRNA molecules. Exemplary antisense and siRNA that reduce theexpression of EGFR are disclosed, e.g., in US 2011/0046067 and Kang, etal. (2006) Cancer Gene Ther. 13(5):530-8. Exemplary miRNA that reducethe expression of EGFR are disclosed, e.g., in U.S. Pat. No. 8,673,872,incorporated herein by reference in its entirety.

An EGFR inhibitor can also be an antibody, antibody fragment, orantibody mimetic that specifically binds EGFR and antagonizes theactivity thereof by, e.g., blocking ligand binding, activation,phosphorylation or protein-protein interactions. Cetuximab (IgG1) andPanitumumab (IgG2) are examples of monoclonal antibody inhibitors ofEGFR. Other antagonistic monoclonal antibodies include Zalutumumab,Nimotuzumab, Matuzumutab, ICR62, and mAb806. See U.S. Pat. Nos.6,506,883, 6,235,883, 5,891,996, 4,943,533, WO 2004/056847, WO2002/092771, WO 2002/66058, and WO 1995/20045. Such antibodies block theextracellular ligand binding domain thereby blocking tyrosine kinaseactivation.

Peptide inhibitors of EGFR that regulate EGFR multimerization andactivation are also of use in this invention. Exemplary peptideinhibitors of EGFR can be based upon the following juxtamembranesequence from EGFR: LLLWALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPS (SEQ IDNO:2), and may optionally include a cell penetrating component such as aprotein transduction domain (PTD) to facilitate delivery into the cell.See US 2016/0311884.

Still further, the EGFR inhibitor can be small molecule that inhibitsthe tyrosine kinase activity of EGFR. Without kinase activity, EGFR isunable to activate itself, which is a prerequisite for binding ofdownstream adaptor proteins. Examples of small molecule inhibitors ofEGFR include, but are not limited to, erlotinib (CAS 183321-74-6),gefitinib (CAS 184475-35-2), lapatinib (CAS 231277-92-2, dual EGFR andERBB2 inhibitor), neratinib (CAS 698387-09-6), canertinib (CAS267243-28-7), vandetanib (CAS 443913-73-3), afatinib (CAS 439081-18-2),AG 1478 (CAS 153436-53-4), TAK-285 (CAS 871026-44-7, dual HER2 and EGFRinhibitor), ARRY334543 (CAS 845272-21-1, dual EGFR phosphorylationinhibitor), Dacomitinib (CAS 1110813-31-4, EGFR and ERBB2 inhibitor),AZD3759 (CAS 1626387-80-1), NT113 (CAS 1398833-56-1, pan-ERBBinhibitor), OSI-420 (Desmethyl Erlotinib, CAS 183321-86-0, EGFRinhibitor), AZD8931 (CAS 848942-61-9, EGFR, HER2 and HER3 inhibitor),AEE788 (CAS 497839-62-9, EGFR, HER2 and VEGFR 1/2 inhibitor), Pelitinib(EKB-569, CAS 257933-82-7, pan-ErbB inhibitor), CUDC-101 (CAS1012054-59-9, EGFR, HER2 and HDAC inhibitor), XL647 (CAS 651031-01-5,dual HER2 and EGFR inhibitor), BMS-599626 (CAS 714971-09-2, dual EGFRand HER2 inhibitor), PKC412 (CAS 120685-11-2, EGFR, PKC, cyclicAMP-dependent protein kinase and S6 kinase inhibitor), BIBX1382 (CAS196612-93-8, EGFR inhibitor) and AP26113 (CAS 1197953-54-0, ALK and EGFRinhibitor), and derivatives and combinations thereof. In someembodiments, the EGFR inhibitor is not Pelitinib.

Ras/Raf/MEK/ERK/MAPK Inhibitors.

Inhibitors of this pathway that selectively decrease or block theexpression of Ras, Raf, MEK, ERK/MAPK include, but are not limited to,antisense, siRNA and miRNA molecules. By way of illustration, antisenseinhibition of MEK1 is disclosed in U.S. Pat. No. 6,096,543, incorporatedherein by reference in its entirety.

Examples of Ras inhibitors include, but are not limited to, R115777 (CAS192185-72-1), BMS-214662 (CAS 195987-41-8), SCH66336 (CAS 193275-84-2),FTI-277 (CAS 1217447-06-7), manumycin A (CAS 52665-74-4), FTI-276 (CAS170006-72-1), RasCAAX (a peptidomimetic), L-744,832 (CAS 1177806-11-9),and derivatives and combinations thereof.

Raf inhibitors of use in this invention include, but are not limited to,Bay43-9006 (sorafenib, CAS 284461-73-0, selective inhibitor for B-Rafand C-Raf), vemurafenib (CAS 918504-65-1, B-Raf inhibitor), dabrafenib(CAS 1195764-45-7, B-Raf inhibitor; see also U.S. Pat. Nos. 7,994,185and 8,415,345), LY3009120 (CAS 1454682-72-4, pan-Raf inhibitor), GW 5074(CAS 220904-83-6, C-Raf-1 inhibitor), ZM 336372 (CAS 208260-29-1, Raf-1inhibitor), 2-bromoaldisine (CAS 96562-96-8, RAF/MEK-1/MAPK pathwayinhibitor), L-779,450 (CAS 303727-31-3), AZ628 (CAS 878739-06-1, Raf-1inhibitor), RAF265 (CAS 927880-90-8, B-Raf and VEGFR-2 inhibitor),encorafenib (LGX818, CAS 1269440-17-6, B-Raf inhibitor), and derivativesand combinations thereof.

MEK inhibitors include, but are not limited to, SL-327 (CAS 305350-87-2,inhibitor of MEK1 and MEK2), PD 184,352 (CAS 212631-79-3),2-bromoaldisine (CAS 96562-96-8, Raf/MEK-1/MAPK pathway inhibitor), PD198306 (CAS 212631-61-3, non-ATP-competitive inhibitor of MEK1/2), PD0325901 (CAS 391210-10-9, inhibitor of MEK and suppressor of ERKphosphorylation), MEK inhibitor II (CAS 623163-52-0), PD 184161 (CAS212631-67-9, selective inhibitor for MEK1 and MEK2), U-0126 (CAS109511-58-2, inhibitor for MEK1/2), PD 98059 (CAS 167869-21-8, selectiveinhibitor for MEK1), AS703026 (CAS 1236699-92-5, inhibitor for MEK1/2),BAY 869766 (CAS 923032-37-5, non-ATP-competitive inhibitor of MEK-1 andMEK-2), PD 318088 (CAS 391210-00-7, inhibitor for MEK1/2), selumetinib(CAS 606143-52-6, MEK-1 non-ATP competitive inhibitor), TAK-733 (CAS1035555-63-5, allosteric inhibitor of MEK), Trametinib (CAS 871700-17-3,allosteric inhibitor of MEK1/MEK2), and derivatives and combinationsthereof. See also WO 1998/037881, WO 1999/901426, WO 2000/041505, WO2000/041994, WO 2000/042002, WO 2000/042003, WO 2000/042022, WO2000/042029, WO 2001/068619, and WO 2002/036570 for additional MEKinhibitors.

ERK inhibitors include, for example, SCH772984 (CAS 942183-800-4, ERK1/2inhibitor), DEL-22379 (CAS 181223-80-3, ERK dimerization inhibitor),VX-11e (CAS 896720-20-0, ERK2 inhibitor), Pluripotin (SC1, CAS839707-37-8, dual ERK1 and RasGAP inhibitor), Ulixertinib (BVD-523,VRT752271, CAS 869886-67-9, ERK1/ERK2 inhibitor), FR 180204 (CAS865362-74-9, ATP-competitive ERK inhibitor), GDC-0994 (CAS 1453848-26-4,ERK1/2 inhibitor), KO-947 (Kura Oncology, ERK1/2 inhibitor), andderivatives and combinations thereof. See also JP 2005-330265 foradditional ERK inhibitors.

JAK/STAT Inhibitors.

Inhibitors that selectively decrease or block the expression of JAK orSTAT include, but are not limited to, antisense, siRNA and miRNAmolecules. By way of illustration, antisense inhibition of STAT-2,STAT-3, STAT-4, STAT-5 and STAT-6 is disclosed in US 2004/0101853, U.S.Pat. Nos. 6,159,694, 6,479,465, 8,722,873 and WO 1998/040478,respectively. Likewise, siRNA for reducing the expression of STAT-1 andSTAT-2 are disclosed in U.S. Pat. No. 9,198,911. STAT-3 siRNA aredescribed in US 2010/0298409, STAT-5 siRNA are described in WO2009/039199, and STAT-6 siRNA are described in U.S. Pat. No. 7,566,700.SiRNA molecules of use in inhibiting the expression of Jak1 and Jak3 aredisclosed in U.S. Pat. No. 9,198,911, incorporated herein by referencein its entirety.

Non-limiting examples of STAT inhibitors include, but are not limitedto, WP-1034 (CAS 857064-42-7, Jak-Stat inhibitor), fludarabine (CAS21679-14-1, STAT1 inhibitor), S3I-201 (CAS 501919-59-1, inhibitor ofSTAT3 DNA-binding activity), Stattic (CAS 19983-44-9, STAT3 inhibitor),APTSTAT3-9R (STAT-binding peptide), STA-21 (CAS 28882-53-3, STAT3inhibitor), SH-4-54 (CAS 1456632-40-8), Napabucasin (CAS 83280-65-3,STAT3 inhibitor), Cryptotanshinone (CAS 35825-57-1, STAT3 inhibitor),niclosamide (CAS 50-65-7, STAT3 inhibitor), NSC 74859 (CAS 501919-59-1,STAT3 inhibitor), HO-3867 (CAS 1172133-28-6, STAT3 inhibitor), andderivatives and combinations thereof.

Jak1/Jak2 inhibitors include, but are not limited to, AG-490 (CAS133550-30-8), CYT387 (CAS 1056634-68-4), SB1518 (Pacritinib, CAS937272-79-2), LY3009104 (INCB28050, Baricitinib, CAS 1187594-09-7),TG101348 (CAS 936091-26-8), BMS-911543 (CAS 1271022-90-2), AZD1480 (CAS935666-88-9), Ruxolitinib (INCB018424, CAS 941678-49-5), CEP-701 (CAS111358-88-4), TG101348 (Fedratinib, CAS 936091-26-8), SD 1008 (CAS960201-81-4, JAK2/STAT3 inhibitor), WP-1066 (CAS 857064-38-1, JAK2/STAT3inhibitor), and derivatives and combinations thereof. JAK3 inhibitorsinclude, but are not limited to, Janex 1 (WHI-P131, CAS 202475-60-3),PF-956980 (CAS 1262832-74-5), WHI-P154 (CAS 211555-04-3), VX-509(Decernotinib, CAS 944842-54-0), JAK3 Inhibitor IV (ZM-39923, CAS1021868-92-7), tofacitinib (CP-690550, CAS 540737-29-9), and derivativesand combinations thereof.

PI3K/AKT/mTOR Inhibitors.

Inhibitors that selectively decrease or block the expression of PI3K,AKT or mTOR include, but are not limited to, antisense, siRNA and miRNAmolecules. By way of illustration, siRNA inhibition of PI3K, AKT andmTOR is disclosed in, e.g., US 2005/0272682, US 2008/0161547, and U.S.Pat. No. 9,012,622 respectively.

Small molecules of use in inhibiting PI3K include, but are not limitedto, SF1101 (LY 294002, CAS 154447-36-6), BKM120 (CAS 944396-07-0),BYL719 (CAS 1217486-61-7), XL-147 (CAS 956958-535), ZSTK-474 (CAS475110-96-4), PX-866 (CAS 502632-66-8), PI-103 (CAS 371935-74-9), andderivatives and combinations thereof.

Exemplary AKT inhibitors include, e.g., AZD5363 (CAS 1143532-39-1),GDC-0068 (CAS 1001264-89-6, ATP-competitive pan-Akt inhibitor), MK-2206(CAS 1032350-13-2), Perifosine (CAS 157716-52-4), PBI-05204 (Oleandrin,CAS 465-16-7), GSK2141795 (CAS 1047634-65-0), and SR13668 (CAS637774-61-9), and derivatives and combinations thereof. Additional AKTinhibitors are described in US 2010/0009397, US 2007/0185152, U.S. Pat.Nos. 6,960,584, 7,098,208, 7,223,738, 7,304,063, 7,378,403, 7,396,832,7,399,764, 7,414,055, 7,544,677, 7,576,209, 7,579,355, 7,589,068,7,638,530, 7,655,649, 7,705,014, 7,750,151, 7,943,732, 8,003,643,8,003,651, 8,008,317, 8,168,652, 8,263,357, 8,273,782, and 8,324,221.

Exemplary dual mTOR/PI3K inhibitors include, e.g., SF1126 (CAS936487-67-1), BEZ235 (CAS 915019-65-7), BGT-226 (CAS 1245537-68-1),PF-04691502 (CAS 1013101-36-4), GNE-477 (CAS 1032754-81-6), XL765 (CAS1349796-36-6), GDC-0941 (CAS 957054-30-7), GDC-0980 (CAS 1032754-93-0),PF-05212384 (CAS 1197160-78-3), and derivatives and combinationsthereof.

Inhibition of mTOR can be achieved using one or more of the followinginhibitors, e.g., OSI-027 (CAS 936890-98-1), INK-128 (CAS 1224844-38-5),AZD-8055 (CAS 1009298-09-2), AZD-2014 (CAS 1009298-59-2), Palomid 529(CAS 914913-88-5), Pp-242 (CAS 1092351-67-1), GSK2126458 (CAS1086062-66-9), PF-04691502 (CAS 1013101-36-4), wortmannin (CAS19545-26-7), Ku-0063794 (CAS 938440-64-3), WAY-600 (CAS 1062159-35-6),WYE-687 (CAS 1062161-90-3), WYE-354 (CAS 1062169-56-5), rapamycin (CAS53123-88-9), and derivatives and combinations thereof. Rapamycinderivatives are further described in, e.g., U.S. Pat. Nos. 5,258,389,5,100,883, 5,118,678, 5,151,413, 5,256,790, 5,120,842, US 2011/0178070,WO 1994/09010, WO 1992/05179, WO 1993/11130, WO 1994/02136, WO1994/02485, WO 1994/02136, WO 1995/16691, WO 1996/41807, WO 1996/41807,WO 1998/02441, WO 2001/14387, and WO 1995/14023. See also US2016/0244424 for additional PI3K/AKT/mTOR inhibitors.

NCK-PAK-JNK Inhibitors.

Inhibitors that selectively decrease or block the expression of NCK, PAKor JNK include, but are not limited to, antisense, siRNA and miRNAmolecules. By way of illustration, siRNA inhibition of Pak1 is disclosedin, e.g., WO 2013/135745. Similarly, siRNA inhibition of JNK1, JNK2 andJNK3 is disclosed in, e.g., US 2015/0361184.

Inhibitors of PAK kinases are known in the art and include, but are notlimited to, 2-aminopyrido[2,3-d]pyrimidin-7(8H)-ones such as thosedisclosed in WO 2009/086204, WO 2010/071846, WO 2011/044535, WO2011/156646, WO 2011/156786, WO 2011/156640, WO 2011/156780, WO2011/156775, and WO 2011/044264; 1H-thieno[3,2-c]pyrazoles,3-amino-tetrahydropyrrolo[3,4-c]pyrazoles andN4-(1H-pyrazol-3-yl)pyrimidine-2,4-diamines as disclosed in WO2004/007504, WO 2007/023382, WO 2007/072153, and WO 2006/072831;N2-bicyclic indolyl, indazolyl and benzimidazolyl derivatives ofN4-(1H-pyrazol-3-yl)pyrimidine-2,4-diamines as described in U.S. Pat.No. 8,637,537; PF-3758309 (CAS 898044-15-0); IPA-3 (CAS 42521-82-4);FRAX597 (CAS 1286739-19-2); FRAX486 (CAS 1232030-35-1); FRAX1036 (CAS1432908-05-8); and derivatives and combinations thereof.

Non-limiting examples of JNK1, JNK2 and/or JNK3 inhibitors include, butare not limited to, JNK Inhibitor V (CAS 345987-15-7), JNK Inhibitor VII(TAT-TI-JIPi₅₃₋₁₆₃, CAS 305350-87-2), JNK Inhibitor VIII (CAS894804-07-0), JNK-IN-7 (CAS 1408064-71-0), JNK Inhibitor IX (CAS312917-14-9), JNK Inhibitor XI (CAS 2207-44-5), JNK Inhibitor XVI (CAS1410880-22-6), AEG 3482 (CAS 63735-71-7), doramapimod (CAS 285983-48-4,p38α MAPK and JNK2 inhibitor), CC-401 (CAS 395104-30-0), SP600125 (CAS129-56-6), AS601245 (CAS 345987-15-7), and derivatives and combinationsthereof. In some embodiments, the inhibitor is not leflunomide.

PLC-DAG-PKC Inhibitors.

Inhibitors that selectively decrease or block the expression of PLC orPKC include, but are not limited to, antisense, siRNA and miRNAmolecules. By way of illustration, siRNA inhibition of PLC is disclosedin U.S. Pat. No. 9,546,367, the siRNA molecules of which areincorporated herein by reference.

Anti-PLCγ antibodies are also known in the art for use in modulating thebinding and/or catalytic activity of a PLCγ. Examples of anti-PLCγantibodies are described in, for example, Lee, et al. (2002) Mol. Vis.8:17-25 and Buckley, et al. (2004) J. Biol. Chem. 279:41807-14.

Examples of small molecule inhibitors of PLC include, but are notlimited to, D609 (CAS 83373-60-8), edelfosine (ET-18-OCH3, CAS77286-66-9, dual PLC/PKC inhibitor), manoalide (CAS 75088-80-1), NCDC(CAS 10556-88-4), U-73122 (CAS 112648-68-7), and derivatives andcombinations thereof.

Her2/Neu Inhibitors.

Inhibitors that selectively decrease or block the expression of Her2/neuinclude, but are not limited to, antisense, siRNA and miRNA molecules.By way of illustration, siRNA inhibition of Her2/neu is disclosed in,e.g., Faltus, et al. (2004) Neoplasia 6(6):786-95; Choudhury, et al.(2004) Int. J. Cancer 108:71-77.

Anti-Her2/neu antibodies are also known in the art for use in modulatingthe activity of Her2/neu. Examples of anti-Her2/neu antibodies include,but are not limited to, trastuzumab (HERCEPTIN, CAS 180288-69-1) andpertuzumab (PERJETA, CAS 380610-27-5). See Schroeder, et al. (2014)Molecules 19:15196-15212 for review.

Examples of small molecule inhibitors of Her2/neu include, but are notlimited to, Lapatinib (TYKERP, CAS 231277-92-2, dual EGFR/Her2inhibitor), Afatinib (GIOTRIF, CAS 439081-18-2, irreversible paninhibitor), AZD8931 (CAS 848942-61-0, EGFR/Her2/ErbB3 inhibitor),AST-1306 (CAS 897383-62-9, irreversible EGFR and Her2 inhibitor),AEE-788 (CAS 497839-62-0, a dual EGFR and Her2 kinase inhibitor),CI-1033 (Canertinib, CAS 289499-45-2, EGFR and Her2 inhibitor), TAK-165(Mubritinib, CAS 366017-09-6, Her2 inhibitor, see U.S. Pat. Nos.6,716,863 and 7,005,526), CP-724714 (CAS 383432-38-0, Her2 inhibitor),CUDC-101 (CAS 1012054-59-9, irreversible HDAC/EGFR/Her2 inhibitor),TAK-285 (CAS 871026-44-7, dual EGFR/Her2 inhibitor), AC-480 (BMS-599626,CAS 714971-09-2, reversible EGFR/Her2/HER4 inhibitor), PF299804 or PF299(Dacomitinib, CAS 1110813-31-4, irreversible EGFR/Her2/Her4 inhibitor),and EKB-569 (Perlitinib, CAS 257933-82-7, dual EGFR/Her2 inhibitor), andderivatives and combinations thereof. In certain embodiments, theinhibitor is selective for Her2 and exhibits little or no activityagainst other kinases.

Aurora Kinase Inhibitors.

Inhibitors that selectively decrease or block the expression of anAurora kinase include, but are not limited to, antisense, siRNA andmiRNA molecules. By way of illustration, siRNA inhibition of Aurorakinase is disclosed in, e.g., Tao, et al. (2007) Br. J. Cancer97(12):1664-1672; Umene, et al. (2015) Int. J. Oncol. 46(4):1498-1506.

Examples of small molecule inhibitors of Aurora kinase include, but arenot limited to, SNS314 Mesylate (CAS 1146618-41-8, pan Aurora inhibitor,see US 2016/0287602, US 2015/0329828 and US 2011/0014191), PHA-680632(CAS 398493-79-3, pan Aurora inhibitor), VE-465 (Tozasertib, VX-680 orMK0457, CAS 639089-54-6), Barasertib (AZD1152, CAS 722544-51-6, Aurora Bkinase inhibitor), Alisertib (MLN8237, CAS 1028486-01-2, Aurora A kinaseinhibitor), Danusertib (PHA-739358, CAS 827318-97-8, pan Aurorainhibitor), PF-03814735 (CAS 942487-16-3, dual Aurora A/B inhibitor),AMG 900 (CAS 945595-80-2, pan Aurora inhibitor), and derivatives andcombinations thereof. In certain embodiments, the inhibitor is selectivefor Aurora kinase and exhibits little or no activity against otherkinases.

PDGFR Inhibitors.

Inhibitors that selectively decrease or block the expression of a PDGFRinclude, but are not limited to, antisense, siRNA and miRNA molecules.By way of illustration, siRNA inhibition of PDGFR is disclosed in, e.g.,Chen, et al. (2008) Liver Int. 28(10):1446-1457; Kaulfuβ, et al. (2013)Oncotarget 4(7):1037-49; Yeh, et al. (2011) BMC Cancer 11:139.

Anti-PDGFR antibodies are also known in the art for use in modulatingthe activity of PDGFR. Examples of anti-PDGFR antibodies include, butare not limited to, IMC-3G3 (anti-PDGFRα antibody; EP 2100618) andIMC-2C5 (PDGFRβ antibody; Shen, et al. (2009) Neoplasia 11(6):594-604).

Examples of small molecule inhibitors of PDGFR include, but are notlimited to, Ki11502 (CAS 347155-76-4), imatinib (GLEEVEC/ST571, CAS220127-57-1, PDGFRα/BCR-ABL/c-kit inhibitor), Ponatinib (AP24534, CAS943319-70-8, Abl/PDGFRα/VEGFR2/FGFR1/Src inhibitor), Telatinib (CAS332012-40-5, VEGFR/c-Kit/PDGFRα inhibitor), Amuvatinib (MP-470, CAS850879-09-3, c-Kit/PDGFRα/Flt3 inhibitor), Crenolanib (CP-868596, CAS670220-88-9, selective inhibitor of PDGFRα/β, see U.S. Pat. Nos.7,071,337, 7,183,414, US 2015/0238479 and US 2010/0016353), Axitinib(CAS 319460-85-0, VEGFR1/VEGFR2/VEGFR3/PDGFRβ/c-Kit inhibitor),CP-673451 (CAS 343787-29-1, inhibitor of PDGFRα/β), Nintedanib (BIBF1120, CAS 656247-17-5, VEGFR/FGFR/PDGFRα/β inhibitor), Masitinib (CAS790299-79-5, Kit/PDGFRα/β inhibitor), Sunitinib (SUTENT/SU11248, CAS557795-19-4, VEGFR2/PDGFRβ inhibitor), TSU-68 (SU6668 or Orantinib, CAS252916-29-3), Linifanib (ABT-869, CAS 796967-16-3, VEGFR/PDGFRinhibitor), AC 710 (CAS 1351522-04-7, selective PDGFR family inhibitor),DMPQ dihydrochloride (CAS 137206-97-4, PDGFRβ inhibitor), GSK 1363089(CAS 849217-64-7, PDGFR/MET/VEGFR2/Ron/AXL inhibitor), PD 166285 (CAS212391-63-4, PDGFβ/FGFR/Src inhibitor) and Toceranib (CAS 356068-94-5,PDGFR and VEGFR inhibitor), and derivatives and combinations thereof.

Atonal-Associated Factor.

Atonal-associated factors are a family of transcription factors thattransdifferentiate supporting cells into sensory hair cells in the ear.Atonal-associated factors are transcription factors of the basichelix-loop-helix (bHLH) family of proteins. The basic domain of theprotein is responsible for DNA binding and function of the protein. TheDrosophila bHLH protein (ato) activates genes associated with thedevelopment of sensory organs of the insect, specifically chordotonalorgans. Atonal-associated factors also referred to as Atonal Homolog 1(Atoh1) proteins are found in a variety of animals and insects,including mice (mouse atonal homolog 1 (Math1)), chickens (chickenatonal homolog 1 (Cath1)), Xenopus (Xenopus atonal homolog 1 (Xath1)),and humans (human atonal homolog 1 (Hath1)). Math1 is highly homologousto ato in the bHLH domain (82% amino acid similarity) with 100%conservation of the basic domain, and functions in determining cell fatein mice. Math1 has been shown to be essential for hair cell developmentand can stimulate hair cell regeneration in the ear. Math1 is furthercharacterized in, for example, Ben-Arie, et al. (1996) Human Mol. Genet.5:1207-1216; Bermingham, et al. (1999) Science 284:1837-1841; Zheng &Gao (2000) Nature Neurosci. 3(2):580-586; and Chen, et al. (2002)Development 129:2495-2505. Hath1 is the human counterpart of Math1. Incertain embodiments, the atonal-associated factor is Math1 or Hath1 or aprotein sharing significant amino acid sequence similarity with that ofMath1 and Hath1. Atonal-associated factors are further described in WO2000/73764.

The amino acid sequence of Hath1 and Math1 are known in the art andavailable under GENBANK Accession Nos. NP_005163 (Gene ID:474) andNP_031526 (Gene ID: 11921), respectively. A protein having significantamino acid sequence similarity with that of Math1 and Hath1 desirablyhas an amino acid sequence that is at least about 50% identical to theamino acid sequence of Hath1 (NP_005163) or Math1 (NP_031526), and hasthe ability to transdifferentiate supporting cells into sensory haircells. Ideally, the Atonal-associated factor has at least about 60%amino acid sequence identity (e.g., at least about 65%, or at leastabout 70%, sequence identity), preferably at least about 75% amino acidsequence identity (e.g., at least about 80%, or at least about 85%,sequence identity), and most preferably at least about 90% amino acidsequence identity (e.g., at least about 95% sequence identity) with theHath1 (NP_005163) or Math1 (NP_031526) amino acid sequence. Looking tothe nucleic acid sequence encoding the Atonal-associated factor,preferably the nucleic acid sequence encodes the Hath1 (NP_005163) orMath1 (NP_031526) amino acid sequence (i.e., the portion of the Hath1 orMath1 genes that encode the Hath1 and Math1 proteins absent theregulatory sequences associated with the gene) or cDNA encoding theHath1 or Math1 protein. Nucleic acid sequences encoding Hath1 and Math1are publicly available under GENBANK Accession Nos. NM_005172 andNM_031526, respectively.

While wild-type Hath1 or Math1 proteins and nucleic acids areparticularly useful, many modifications and variations (e.g., mutation)of the Hath1 or Math1 sequences are possible and appropriate in thecontext of the invention. For example, the degeneracy of the geneticcode allows for the substitution of nucleotides throughout the codingsequence, as well as in the translational stop signal, withoutalteration of the encoded polypeptide. Such substitutions can be deducedfrom the known amino acid sequence of an atonal-associated factor ornucleic acid sequence encoding an atonal-associated factor and can beconstructed by conventional synthetic or site-specific mutagenesisprocedures. Synthetic DNA methods can be carried out in accordance withthe procedures of Itakura, et al. (1977) Science 198:1056-1063 or Crea,et al. (1978) Proc. Natl. Acad. Sci. USA 75:5765-5769. Site-specificmutagenesis procedures are described in Maniatis, et al. (1989)Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor,NY Alternatively, the nucleic acid sequence can encode anatonal-associated peptide with extensions on either the N- or C-terminusof the protein, so long as the resulting atonal-associated factorretains activity (i.e., the ability to transdifferentiate supportingcells into sensory hair cells).

The function of atonal-associated factors is dependent on thehelix-loop-helix (HLH) portion of the protein, particularly the basicregion of the HLH domain (Chien, et al. (1996) Proc. Natl. Acad. Sci.93:13239-13244), which includes the amino acid sequenceAANARERRRMHGLNHAFDQLR (SEQ ID NO:1). Accordingly, any modification ofthe atonal-associated factor amino acid sequence desirably is locatedoutside of the basic domain of the protein. Exemplary constructs andexpression vectors harboring nucleic acids encoding atonal-associatedfactor are provided WO 2004/076626.

Sensory Perception.

The invention provides for the modulation of sensory perception in ananimal by administering to the inner ear an inhibitor of EGFR signalingand optionally an expression vector (e.g., expression viral vector)harboring a nucleic acid molecule encoding an atonal-associated factorand/or otoprotective agent. By “modulating sensory perception” it ismeant achieving, at least in part, the ability to recognize and adapt toenvironmental changes. In terms of sensory hair cell function,modulation in sensory perception is associated with the generation orprotection of sensory hair cells that convert mechanical stimuli in theinner ear into neural impulses, which are then processed in the brainsuch that an animal is aware of environmental change, e.g., sound,language, or body/head position. Sensory hair cells are preferablygenerated in the organ of Corti and/or vestibular apparatus. In thecontext of prophylaxis, sensory hair cells, which would otherwise beinitially or further damaged or lost due to, e.g., ototoxic agents, areprotected from damage or loss by the administration of an inhibitor ofEGFR signaling and optionally an otoprotective agent. In the context oftreatment, the combination of the inhibitor of EGFR signaling and thenucleic acid molecule for expressing an atonal-associated factorincreases reprogramming efficiency and/or terminal differentiation ofsome reprogrammed cells thereby facilitating the generation of sensoryhair cells that allow perception (or recognition) of stimuli in theinner ear.

Sensory hair cell generation can be determined using a variety of means,such as those known to one skilled in the art. Hair cells can bedetected via scanning electron microscopy and/or via detection of myosinVIIa, a hair cell-specific protein detected by immunochemistry. However,the mere presence of sensory hair cells does not necessarily imply afunctional system for recognizing environmental stimuli. Functionalsensory hair cells must be operably linked to neural pathways, such thatmechanical stimuli are translated to nerve impulses recognized by thebrain. Accordingly, while detection of hair cell generation isappropriate for determining successful expression of theatonal-associated nucleic acid sequence to target tissue, examination ofsubject awareness is a more desirable indicator of changes in sensoryperception.

A change in the ability of a subject to detect sound is readilyaccomplished through administration of simple hearing tests, such as atone test commonly administered by an audiologist. In most mammals, areaction to different frequencies indicates a change in sensoryperception. In humans, comprehension of language also is appropriate.For example, it is possible for a subject to hear while being unable tounderstand speech. A change in perception is indicated by the ability todistinguish different types of acoustic stimuli, such as differentiatinglanguage from background noise, and by understanding speech. Speechthreshold and discrimination tests are useful for such evaluations.

Evaluation of changes in balance, motion awareness, and/or timing ofresponse to motion stimuli also is achieved using a variety oftechniques. Vestibular function also can be measured by comparing themagnitude of response to motion stimulus (gain) or timing of initiationof response (phase). Animals can be tested for Vestibulo-Ocular Reflex(VOR) gain and phase using scleral search coils to evaluate improvementsin sensory perception. Electronystagmography (ENG) records eye movementsin response to stimuli such as, for instance, moving or flashing lights,body repositioning, fluid movement inside the semicircular canals, andthe like. Evaluation of balance during movement using a rotating chairor moving platform also is useful in this respect.

To detect a change in sensory perception, a baseline value is recordedprior to the inventive method using any appropriate sensory test. Asubject is reevaluated at an appropriate time period following theinventive method (e.g., 1 hour, 6 hours, 12 hours, 18 hours, 1 day, 3days, days, 7 days, 14 days, 21 days, 28 days, 2 months, 3 months ormore following the inventive method), the results of which are comparedto baseline results to determine a change in sensory perception.

Method of Prevention or Treatment.

The inventive method promotes the protection and/or generation ofsensory hair cells that allow perception of stimuli. Accordingly, thisinvention provides a method for the prevention, treatment, control,amelioration, or reduction of risk of hearing impairments, loss anddisorders by administering to a subject in need of treatment aninhibitor of EGFR signaling and optionally an expression vector (e.g.,expression viral vector) harboring a nucleic acid molecule encoding anatonal-associated factor and/or one or more otoprotective/regenerativeagents. Ideally, the inventive method prophylactically ortherapeutically treats an animal for at least one disorder associatedwith loss, damage, absence of sensory hair cells, such as hearing lossand balance disorders. Hearing loss can be caused by damage of haircells of the organ of Corti due to bacterial or viral infection,heredity, physical injury, acoustic trauma, ototoxic drugs (e.g.,aminoglycoside antibiotic or cisplatin) and the like. While hearing lossis easily identified, balance disorders manifest in a broad variety ofcomplications easily attributable to other ailments. Symptoms of abalance disorder include disorientation, dizziness, vertigo, nausea,blurred vision, clumsiness, and frequent falls. Balance disorderstreated by the inventive method preferably involve a peripheralvestibular disorder (i.e., a disturbance in the vestibular apparatus)involving dysfunctional translation of mechanical stimuli into neuralimpulses due to damage or lack of sensory hair cells.

In one aspect, methods of protecting against or preventing hearing lossor impairment are provided. In accordance with such methods, a subjectin need of treatment is administered an effective amount of inhibitor ofEGFR signaling. In some embodiments, the inhibitor of EGFR signalinginhibits the expression or activity of EGFR, a Ras/Raf/MEK/ERK/MAPKprotein, a JAK/STAT protein, a PI3K/AKT/mTOR protein, a NCK-PAK-JNKprotein, a PLC-DAG-PKC protein, or a cell cycle-associated proteinkinase associated with or downstream of EGFR. In other embodiments,prevention of hearing loss is achieved by administering to a subject inneed of treatment an inhibitor of a cell cycle-associated protein kinaseassociated with or downstream of EGFR. In certain embodiments,prevention of hearing loss is achieved by administering to a subject inneed of treatment an inhibitor of Her-2, Aurora kinase, B-Raf, or PDGFRexpression or activity. The inhibitor of EGFR signaling can beadministered alone or in combination with one or more otoprotectiveagents. The term “otoprotective agent” refers to an agent that reducesor prevents noise-induced hearing loss, chemically-induced hearing loss,or age-induced hearing impairment or otherwise protects against hearingimpairment. Examples of otoprotective agents include, but are notlimited to, PARP-1 inhibitors; pirenzepine LS-75, otenzepad, AQ-RA741,viramune, BIBN 99, DIBD, telenzepine (see US 2011/0263574); methionine(see U.S. Pat. No. 7,071,230); IGF-1, FGF-2, aspirin, reducedglutathione, N-methyl-(D)-glucaminedithiocarbamate, and iron chelatorssuch as tartrate and maleate. See also US 2005/0101534 for additionalotoprotective agents.

In another aspect, methods of treating hearing loss or impairment areprovided. In accordance with such methods, a subject in need oftreatment is administered an effective amount of inhibitor of EGFRsignaling in combination with an expression vector harboring a nucleicacid molecule for expressing an atonal-associated factor. In someembodiments, the inhibitor of EGFR signaling inhibits the expression oractivity of EGFR, a Ras/Raf/MEK/ERK/MAPK protein, a JAK/STAT protein, aPI3K/AKT/mTOR protein, a NCK-PAK-JNK protein, a PLC-DAG-PKC protein, ora cell cycle-associated protein kinase associated with or downstream ofEGFR. In other embodiments, treatment of hearing loss is achieved byadministering to a subject in need of treatment an inhibitor of EGFR, aRas/Raf/MEK/ERK/MAPK protein, a JAK/STAT protein, a PI3K/AKT/mTORprotein, a NCK-PAK-JNK protein, or a PLC-DAG-PKC protein. In certainembodiments, treatment of hearing loss is achieved by administering to asubject in need of treatment an inhibitor of EGFR, PLC, STAT3, JAK2,PI3K or MEK expression or activity. The inhibitor of EGFR signaling andexpression vector harboring a nucleic acid molecule for expressing anatonal-associated factor can be administered alone or in combinationwith one or more regenerative agents. The term “regenerative agent”refers to an agent that stimulates or promotes sensory hair cellregeneration. Examples of regenerative agents include, but are notlimited to, nicotinamide riboside (see US 2015/0174148); siRNA targetingHes1 (see U.S. Pat. No. 9,101,647); PKA inhibitors (see U.S. Pat. No.6,268,351); a Myc family protein such as c-Myc, N-Myc or L-Myc (see US2015/0079110); and ellipticine derivatives and/or a GSK-3 inhibitor (seeU.S. Pat. No. 9,370,510).

Protection against and prevention or treatment of hearing loss orimpairment can be in the context of conditions including, but notlimited to, tinnitus, ringing, Presbyacusis, auditory neuropathy,acoustic trauma, acoustic neuroma, Pendred syndrome, Usher syndrome,Wardenburg syndrome, non-syndromic sensorineural deafness, otitis media,otosclerosis, Meniere's disease, ototoxicity, labyrinthitis, as well ashearing impairments caused by infection (i.e., measles, mumps, ormeningitis), medicines such as antibiotics, and some cancer treatments(i.e., chemotherapy and radiation therapy).

In certain embodiments, the hearing impairment is drug-induced. In astill further aspect, the drug is a chemotherapeutic agent. Morespecifically, the drug is a platinum-based chemotherapeutic agent suchas carboplatin, cisplatin, transplatin, nedaplatin, oxaliplatin,picoplatin, satraplatin, transplatin, and triplatin, or apharmaceutically acceptable salt thereof. In a particular embodiment,the platinum-based chemotherapeutic agent is cisplatin, or apharmaceutically acceptable salt thereof. In yet a further embodiment,the drug is an antibiotic, including, but not limited to, daunorubicin,doxorubicin, epirubicin, idarubicin, actinomycin-D, bleomycin,mitomycin-C, amikacin, apramycin, arbekacin, astromicin, bekanamycin,dibekacin, framycetin, gentamicin, hygromycin B, isepamicin, kanamycin,neomycin, netilmicin, paromomycin, rhodostreptomycin, ribostamycin,sisomicin, spectinomycin, streptomycin, tobramycin, and verdamicin, or apharmaceutically acceptable salt thereof.

In a further aspect, the hearing impairment is age-related,noise-induced or a balance or orientation-related disorder. Examples ofbalance disorders include, but are not limited to, induced orspontaneous vertigo, dysequilibrium, increased susceptibility to motionsickness, nausea, vomiting, ataxia, labyrinthitis, oscillopsia,nystagmus, syncope, lightheadedness, dizziness, increased falling,difficulty walking at night, Meniere's disease, and difficulty in visualtracking and processing. Further, the noise-induced hearing loss may betemporary or permanent.

More than one billion teens and young adults worldwide are at risk ofhearing loss from exposure to loud music, as recently reported by theWorld Health Organization. Many other noise exposures, includingoccupational settings and consumer-operated devices, also causenoise-induced hearing loss, which is among the most common physicalcomplaints and which detracts significantly from the ability toconverse, communicate, and participate in everyday life (thus reducinggeneral quality of life of the individual and the family). Acute orchronic acoustic overexposure has put more than 40 million US workers atrisk of permanent hearing loss (Kopke, et al. (2007) Hear. Res.226:114-125).

Traumatic brain injury (TBI) and blast-associated injury occur mostfrequently in military situations where blast exposure cannot bepredicted, trauma intensity exceeds the effectiveness of protectivedevices, or protective devices are not available. TBI is oftenaccompanied by a diverse range of disruption or damage to the auditorysensory system, which is highly vulnerable to blast injury. Extremephysical blast force can cause damage of various types to the peripheralauditory system, including rupture of the tympanic membrane (TM,eardrum), fracture of the middle ear bones, dislocation of sensory haircells from the basilar membrane, and loss of spiral ganglia thatinnervate hair cells. In human studies of blast injury, approximately17-29% of cases involve severe TM rupture, while 33-78% involve moderateto severe sensorineural hearing loss (hair cell and ganglion loss).Therefore, TBI and blast injury are a common, although extreme, cause ofhearing loss.

Biological protection of hearing is more promising than currentlyavailable mechanical protective devices. Hearing aids are frequentlyproblematic because of their high cost and their many technical issues.Ideally, service men and women could take protective drugs beforeentering high-risk or high-noise settings and would then be protectedfrom noise injury with no effect on performance. To date, there are noFDA-approved drugs for protection against noise- and TBI-associatedhearing loss.

In accordance with the methods of this invention, the inhibitor of EGFRsignaling and optional expression vector harboring a nucleic acidmolecule for expressing an atonal-associated factor can be administeredlocally, e.g., to the inner ear of the subject. Alternatively, theinhibitor of EGFR signaling and optional expression vector harboring anucleic acid molecule for expressing an atonal-associated factor can beadministered systemically. Further, the inhibitor of EGFR signaling andoptional expression vector harboring a nucleic acid molecule forexpressing an atonal-associated factor can be administered via injectioninto one or more of the scala tympani, cochlear duct, scala vestibule ofthe cochlea, into the auditory nerve trunk in the internal auditorymeatus, or into the middle ear space across the transtympanicmembrane/ear drum. Moreover, when used in combination, the EGFRsignaling and expression vector harboring a nucleic acid molecule forexpressing an atonal-associated factor can be administered via the sameor different routes.

In various aspects, the disclosed molecules can be used in combinationwith one or more other drugs in the treatment, prevention, control,amelioration, or reduction of risk of hearing impairments and disordersfor which disclosed molecules or the other drugs can have utility, wherethe combination of the drugs together are safer or more effective thaneither drug alone. Such other drug(s) can be administered, by a routeand in an amount commonly used therefor, contemporaneously orsequentially with a compound of the present invention. When a moleculeof the present invention is used contemporaneously with one or moreother drugs, a pharmaceutical composition in unit dosage form containingsuch other drugs and a disclosed compound is preferred. However, thecombination therapy can also include therapies in which a disclosedmolecule and one or more other drugs are administered on differentoverlapping schedules. It is also contemplated that when used incombination with one or more other active ingredients, the disclosedmolecules and the other active ingredients can be used in lower dosesthan when each is used singly.

The methods herein are useful in the prevention or treatment of bothacute and persistent, progressive disorders associated with lack of ordamage to functional sensory hair cells. For acute ailments, the drugsherein can be administered using a single application or multipleapplications within a short time period. For persistent diseases, suchas hearing loss, or disorders stemming from a massive loss of sensoryhair cells, numerous rounds of administration of the drugs herein may benecessary to realize a therapeutic effect.

Where appropriate, following treatment, the subject (e.g., human orother animal) can be tested for an improvement in hearing or in othersymptoms related to hearing disorders. Subjects benefiting fromtreatment include those at risk of hair cell loss and/or a patient withhair cell loss. For example, a subject having or at risk for developinga hearing loss can hear less well than the average subject (e.g., anaverage human being), or less well than a subject before experiencingthe hearing loss. For example, hearing can be diminished by at least 5%,10%, 30%, 50% or more. Methods for measuring hearing are well-known andinclude pure tone audiometry, air conduction, and bone conduction tests.These exams measure the limits of loudness (intensity) and pitch(frequency) that a human can hear. Hearing tests in humans includebehavioral observation audiometry (for infants to seven months), visualreinforcement orientation audiometry (for children 7 months to 3 years)and play audiometry for children older than 3 years. Oto-acousticemission testing can be used to test the functioning of the cochlearhair cells, and electro-cochleography provides information about thefunctioning of the cochlea and the first part of the nerve pathway tothe brain. In various aspects, treatment can be continued with orwithout modification or can be stopped.

In various aspects, the methods described herein can be used to generatehair cell growth in the ear and/or to increase the number of hair cellsin the ear (e.g., in the inner, middle, and/or outer ear). In thisrespect, an effective amount of a molecule described herein is an amountthat increases the number of hair cells in the ear by about 2-, 3-, 4-,6-, 8-, or 10-fold, or more, as compared to the number of hair cellsbefore treatment. This new hair cell growth can effectively restore orestablish at least a partial improvement in the subject's ability tohear. For example, administration of a stimulatory agent and aninhibitory agent of this invention can improve hearing loss by about 5,10, 15, 20, 40, 60, 80, 100% or more.

Expression Vectors.

One of ordinary skill in the art will appreciate that any of a number ofexpression vectors known in the art are suitable for introducing anucleic acid sequence to the inner ear. Examples of suitable expressionvectors include, for instance, plasmids, plasmid-liposome complexes, andviral vectors, e.g., parvoviral-based vectors (i.e., adeno-associatedvirus (AAV)-based vectors), retroviral vectors, herpes simplex virus(HSV)-based vectors, AAV-adenoviral chimeric vectors, andadenovirus-based vectors. Any of these expression vectors can beprepared using standard recombinant DNA techniques described in, e.g.,Sambrook, et al. (1989) Molecular Cloning, A Laboratory Manual, 2^(nd)Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY; and Ausubel, etal. (1994) Current Protocols in Molecular Biology, Greene PublishingAssociates and John Wiley & Sons, New York, NY.

Plasmids, genetically engineered circular double-stranded DNA molecules,can be designed to contain an expression cassette for delivery of anucleic acid sequence to the inner ear. Although plasmids were the firstvector described for the administration of therapeutic nucleic acids,the level of transfection efficiency is poor compared with othertechniques. By complexing the plasmid with liposomes, the efficiency ofgene transfer in general is improved. While the liposomes used forplasmid-mediated gene transfer strategies have various compositions,they are typically synthetic cationic lipids. Advantages ofplasmid-liposome complexes include their ability to transfer largepieces of DNA encoding a therapeutic nucleic acid and their relativelylow immunogenicity. Plasmids also can be modified to prolong transgeneexpression as described in U.S. Pat. No. 6,165,754. Expression of atransgene in the ear using plasmids has been described (see, forexample, Jero, et al. (2001) Human Gene Ther. 12:539-549). Whileplasmids are suitable for use in the inventive method, preferably theexpression vector is a viral vector.

AAV vectors are viral vectors of particular interest for use in genetherapy protocols. AAV is a DNA virus, which is not known to cause humandisease. AAV requires co-infection with a helper virus (i.e., anadenovirus or a herpes virus), or expression of helper genes, forefficient replication. AAV vectors used for administration of atherapeutic nucleic acid have approximately 96% of the parental genomedeleted, such that only the terminal repeats (ITRs), which containrecognition signals for DNA replication and packaging, remain. Thiseliminates immunologic or toxic side effects due to expression of viralgenes. Host cells containing an integrated AAV genome show no change incell growth or morphology (see, for example, U.S. Pat. No. 4,797,368).Although efficient, the need for helper virus or helper genes can be anobstacle for widespread use of this vector.

Retrovirus is an RNA virus capable of infecting wide variety of hostcells. Upon infection, the retroviral genome integrates into the genomeof its host cell and is replicated along with host cell DNA, therebyconstantly producing viral RNA and any nucleic acid sequenceincorporated into the retroviral genome. When employing pathogenicretroviruses, e.g., human immunodeficiency virus (HIV) or human T-celllymphotrophic viruses (HTLV), care must be taken in altering the viralgenome to eliminate toxicity. A retroviral vector can additionally bemanipulated to render the virus replication-incompetent. As such,retroviral vectors are thought to be particularly useful for stable genetransfer in vivo. Lentiviral vectors, such as HIV-based vectors, areexemplary of retroviral vectors used for gene delivery. Unlike otherretroviruses, HIV-based vectors are known to incorporate their passengergenes into non-dividing cells and, therefore, are particularly useful inthe sensory epithelium of the inner ear where sensory cells do notregenerate.

HSV-based viral vectors are suitable for use as an expression vector tointroduce nucleic acids into the inner ear for transduction of targetcells. The mature HSV virion is composed of an enveloped icosahedralcapsid with a viral genome composed of a linear double-stranded DNAmolecule that is 152 kb. Most replication-deficient HSV vectors containa deletion to remove one or more intermediate-early genes to preventreplication. Advantages of the herpes vector are its ability to enter alatent stage that can result in long-term DNA expression, and its largeviral DNA genome that can accommodate exogenous DNA up to 25 kb. Ofcourse, this ability is also a disadvantage in terms of short-termtreatment regimens. For a description of HSV-based vectors appropriatefor use in the inventive methods, see, for example, U.S. Pat. Nos.5,837,532, 5,846,782, 5,849,572, 5,804,413, WO 1991/02788, WO1996/04394, WO 1998/15637, and WO 1999/06583.

Adenovirus (Ad) is a 36-kb double-stranded DNA virus that efficientlytransfers DNA in vivo to a variety of different target cell types. Foruse in the inventive method, the virus is preferably madereplication-deficient by deleting select genes required for viralreplication. The expendable non-replication-essential E3 region is alsofrequently deleted to allow additional room for a larger DNA insert. Thevector can be produced in high titers and can efficiently transfer DNAto replicating and non-replicating cells. Genetic informationtransferred to a cell by way of an adenoviral vector remainsepi-chromosomal, thus eliminating the risks of random insertionalmutagenesis and permanent alteration of the genotype of the target cell.However, if desired, the integrative properties of AAV can be conferredto adenovirus by constructing an AAV-Ad chimeric vector. For example,the AAV inverted terminal repeats (ITRs) and nucleic acid encoding theRep protein incorporated into an adenoviral vector enables theadenoviral vector to integrate into a mammalian cell genome. Therefore,AAV-Ad chimeric vectors are an interesting option for use in the contextof the invention.

Preferably, the expression vector of the inventive method is a viralvector, more preferably, the expression vector is an adenoviral vector.Adenovirus from any origin, any subtype, mixture of subtypes, or anychimeric adenovirus can be used as the source of the viral genome forthe adenoviral vector of the invention. A human adenovirus preferably isused as the source of the viral genome for the replication-deficientadenoviral vector. The adenovirus can be of any subgroup or serotype.For instance, an adenovirus can be of subgroup A (e.g., serotypes 12,18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35,and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g.,serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40and 41), an unclassified serogroup (e.g., serotypes 49 and 51), or anyother adenoviral serotype. Adenoviral serotypes 1 through 51 areavailable from the American Type Culture Collection (ATCC, Manassas,VA). Preferably, the adenoviral vector is of subgroup C, especiallyserotype 2 or even more desirably serotype 5.

However, non-group C adenoviruses, and even non-human adenoviruses, canbe used to prepare replication-deficient adenoviral gene transfervectors for delivery of DNA to target cells in the inner ear. Preferredadenoviruses used in the construction of non-group C adenoviral genetransfer vectors include Ad12 (group A), Ad7 and Ad35 (group B), Ad30and Ad36 (group D), Ad4 (group E), and Ad41 (group F). Non-group Cadenoviral vectors, methods of producing non-group C adenoviral vectors,and methods of using non-group C adenoviral vectors are disclosed in,for example, U.S. Pat. Nos. 5,801,030, 5,837,511, 5,849,561, WO1997/12986 and WO 1998/53087. Preferred non-human adenoviruses include,but are not limited to, simian (e.g., SAV 25), bovine, canine, porcineadenoviruses.

The adenoviral vector is preferably replication-deficient. By“replication-deficient” is meant that the adenoviral vector comprises anadenoviral genome that lacks at least one replication-essential genefunction (i.e., such that the adenoviral vector does not replicate intypical host cells, especially those in the human patient that could beinfected by the adenoviral vector in the course of treatment inaccordance with the invention). A deficiency in a gene, gene function,or gene or genomic region, as used herein, is defined as a deletion ofsufficient genetic material of the viral genome to impair or obliteratethe function of the gene whose nucleic acid sequence was deleted inwhole or in part. While deletion of genetic material is preferred,mutation of genetic material by addition or substitute also isappropriate for disrupting gene function. Replication-essential genefunctions are those gene functions that are required for replication(e.g., propagation) and are encoded by, for example, the adenoviralearly regions (e.g., the E1, E2, and E4 regions), late regions (e.g.,the L1-L5 regions), genes involved in viral packaging (e.g., the IVa2gene), and virus-associated RNAs (e.g., VA-RNA1 and/or VA-RNA-2). Morepreferably, the replication-deficient adenoviral vector comprises anadenoviral genome deficient in at least one replication-essential genefunction of one or more regions of the adenoviral genome. Preferably,the adenoviral vector is deficient in at least one gene function of theE1 region or the E4 region of the adenoviral genome required for viralreplication (denoted an E1-deficient adenoviral vector or anE4-deficient adenoviral vector). In addition to a deficiency in the E1region, the recombinant adenovirus also can have a mutation in the majorlate promoter (MLP), as discussed in WO 2000/00628. Most preferably, theadenoviral vector is deficient in at least one replication-essentialgene function (desirably all replication-essential gene functions) ofthe E1 region and at least part of the nonessential E3 region (e.g., anXbaI deletion of the E3 region) (denoted an E1/E3-deficient adenoviralvector). With respect to the E1 region, the adenoviral vector can bedeficient in part or all of the E1A region and part or all of the E1Bregion, e.g., in at least one replication-essential gene function ofeach of the E1A and E1B regions. When the adenoviral vector is deficientin at least one replication-essential gene function in one region of theadenoviral genome (e.g., an E1- or E1/E3-deficient adenoviral vector),the adenoviral vector is referred to as “singly replication-deficient.”

The adenoviral vector of the invention can be “multiplyreplication-deficient,” meaning that the adenoviral vector is deficientin one or more replication-essential gene functions in each of two ormore regions of the adenoviral genome. For example, the aforementionedE1-deficient or E1/E3-deficient adenoviral vector can be furtherdeficient in at least one replication-essential gene function of the E4region (denoted an E1/E4- or E1/E3/E4-deficient adenoviral vector),and/or the E2 region (denoted an E1/E2- or E1/E2/E3-deficient adenoviralvector), preferably the E2A region (denoted an E1/E2A- orE1/E2A/E3-deficient adenoviral vector). Ideally, the adenoviral vectorlacks replication-essential gene functions of only thosereplication-essential gene functions encoded by the early regions of theadenoviral genome, although this is not required in all contexts of theinvention. A preferred multiply-deficient adenoviral vector comprises anadenoviral genome having deletions of nucleotides 457-3332 of the E1region, nucleotides 28593-30470 of the E3 region, nucleotides32826-35561 of the E4 region, and, optionally, nucleotides 10594-10595of the region encoding VA-RNA1. However, other deletions may beappropriate. Nucleotides 356-3329 or 356-3510 can be removed to create adeficiency in replication-essential E1 gene functions. Nucleotides28594-30469 can be deleted from the E3 region of the adenoviral genome.While the specific nucleotide designations recited above correspond tothe adenoviral serotype 5 genome, the corresponding nucleotides fornon-serotype 5 adenoviral genomes can easily be determined by those ofordinary skill in the art.

The adenoviral vector, when multiply replication-deficient, especiallyin replication-essential gene functions of the E1 and E4 regions,preferably includes a spacer element to provide viral growth in acomplementing cell line similar to that achieved by singlyreplication-deficient adenoviral vectors, particularly an E1-deficientadenoviral vector. The spacer element can contain any sequence orsequences which are of a desired length, such as sequences at leastabout 15 base pairs (e.g., between about 15 base pairs and about 12,000base pairs), preferably about 100 base pairs to about 10,000 base pairs,more preferably about 500 base pairs to about 8,000 base pairs, evenmore preferably about 1,500 base pairs to about 6,000 base pairs, andmost preferably about 2,000 to about 3,000 base pairs in length. Thespacer element sequence can be coding or non-coding and native ornon-native with respect to the adenoviral genome, but does not restorethe replication-essential function to the deficient region. The use of aspacer in an adenoviral vector is described in U.S. Pat. No. 5,851,806.In one embodiment of the inventive method, the replication-deficient orconditionally-replicating adenoviral vector is an E1/E4-deficientadenoviral vector wherein the L5 fiber region is retained, and a spaceris located between the L5 fiber region and the right-side ITR. Morepreferably, in such an adenoviral vector, the E4 polyadenylationsequence alone or, most preferably, in combination with anothersequence, exists between the L5 fiber region and the right-side ITR, soas to sufficiently separate the retained L5 fiber region from theright-side ITR, such that viral production of such a vector approachesthat of a singly replication-deficient adenoviral vector, particularlyan E1-deficient adenoviral vector.

The adenoviral vector can be deficient in replication-essential genefunctions of only the early regions of the adenoviral genome, only thelate regions of the adenoviral genome, and both the early and lateregions of the adenoviral genome. The adenoviral vector also can haveessentially the entire adenoviral genome removed, in which case it ispreferred that at least either the viral ITRs and one or more promotersor the viral ITRs and a packaging signal are left intact (i.e., anadenoviral amplicon). The 5′ or 3′ regions of the adenoviral genomecomprising ITRs and packaging sequence need not originate from the sameadenoviral serotype as the remainder of the viral genome. For example,the 5′ region of an adenoviral serotype 5 genome (i.e., the region ofthe genome 5′ to the adenoviral E1 region) can be replaced with thecorresponding region of an adenoviral serotype 2 genome (e.g., the Ad5genome region 5′ to the E1 region of the adenoviral genome is replacedwith nucleotides 1-456 of the Ad2 genome). Suitablereplication-deficient adenoviral vectors, including multiplyreplication-deficient adenoviral vectors, are disclosed in U.S. Pat.Nos. 5,837,511, 5,851,806, 5,994,106, US 2001/0043922, US 2002/0004040,US 2002/0031831, US 2002/0110545, WO 1995/34671, WO 1997/12986, and WO1997/21826. Ideally, the replication-deficient adenoviral vector ispresent in a pharmaceutical composition virtually free ofreplication-competent adenovirus (RCA) contamination (e.g., thepharmaceutical composition comprises less than about 1% of RCAcontamination). Most desirably, the pharmaceutical composition isRCA-free. Adenoviral vector compositions and stocks that are RCA-freeare described in U.S. Pat. Nos. 5,944,106, 6,482,616, US 2002/0110545and WO 1995/34671.

Therefore, in a preferred embodiment, the expression vector of theinventive method is a multiply replication-deficient adenoviral vectorlacking all or part of the E1 region, all or part of the E3 region, allor part of the E4 region, and, optionally, all or part of the E2 region.It is believed that multiply deficient vectors are particularly suitedfor delivery of exogenous nucleic acid sequences to the ear. Adenoviralvectors deficient in at least one replication-essential gene function ofthe E1 region are most commonly used for gene transfer in vivo. However,currently used singly replication-deficient adenoviral vectors can bedetrimental to the sensitive cells of the epithelium of the inner ear,causing damage to the very cells to be treated. Adenoviral vectors thatare deficient in at least one replication-essential gene function of theE4 region, particularly adenoviral vectors deficient inreplication-essential gene functions of the E4 region and the E1 region,are less toxic to cells than E1-deficient adenoviral vectors (see, forexample, Wang, et al. (1996) Nature Med. 2(6):714-716 and U.S. Pat. No.6,228,646). Accordingly, damage to existing hair cells and supportingcells can be minimized by employing an E1,E4-deficient adenoviral vectorto deliver the nucleic acid sequence encoding the atonal-associatedfactor to inner ear cells.

In this regard, it has been observed that an at least E4-deficientadenoviral vector expresses a transgene at high levels for a limitedamount of time in vivo and that persistence of expression of a transgenein an at least E4-deficient adenoviral vector can be modulated throughthe action of a trans-acting factor, such as HSV ICPO, Ad pTP, CMV-IE2,CMV-IE86, HIV tat, HTLV-tax, HBV-X, AAV Rep 78, the cellular factor fromthe U205 osteosarcoma cell line that functions like HSV ICPO, or thecellular factor in PC12 cells that is induced by nerve growth factor,among others. In view of the above, the multiply deficient adenoviralvector (e.g., the at least E4-deficient adenoviral vector) or a secondexpression vector comprises a nucleic acid sequence encoding atrans-acting factor that modulates the persistence of expression of thenucleic acid sequence encoding the atonal-associated factor, asdescribed in, for example, U.S. Pat. Nos. 6,225,113, 6,660,521,6,649,373, and WO 2000/34496.

Replication-deficient adenoviral vectors are typically produced incomplementing cell lines that provide gene functions not present in thereplication-deficient adenoviral vectors, but required for viralpropagation, at appropriate levels in order to generate high titers ofviral vector stock. A preferred cell line complements for at least oneand preferably all replication-essential gene functions not present in areplication-deficient adenovirus. The complementing cell line cancomplement for a deficiency in at least one replication-essential genefunction encoded by the early regions, late regions, viral packagingregions, virus-associated RNA regions, or combinations thereof,including all adenoviral functions (e.g., to enable propagation ofadenoviral amplicons). Most preferably, the complementing cell linecomplements for a deficiency in at least one replication-essential genefunction (e.g., two or more replication-essential gene functions) of theE1 region of the adenoviral genome, particularly a deficiency in areplication-essential gene function of each of the E1A and E1B regions.In addition, the complementing cell line can complement for a deficiencyin at least one replication-essential gene function of the E2(particularly as concerns the adenoviral DNA polymerase and terminalprotein) and/or E4 regions of the adenoviral genome. Desirably, a cellthat complements for a deficiency in the E4 region comprises the E4-ORF6gene sequence and produces the E4-ORF6 protein. Such a cell desirablycomprises at least ORF6 and no other ORF of the E4 region of theadenoviral genome. The cell line preferably is further characterized inthat it contains the complementing genes in a non-overlapping fashionwith the adenoviral vector, which minimizes, and practically eliminates,the possibility of the vector genome recombining with the cellular DNA.Accordingly, the presence of replication competent adenoviruses (RCA) isminimized if not avoided in the vector stock, which, therefore, issuitable for certain therapeutic purposes, especially gene therapypurposes. The lack of RCA in the vector stock avoids the replication ofthe adenoviral vector in non-complementing cells. Construction of such acomplementing cell lines involve standard molecular biology and cellculture techniques, such as those described by Sambrook, et al. (1989)Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring HarborPress, Cold Spring Harbor, NY; and Ausubel, et al. (1994) CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, New York, NY.

Complementing cell lines for producing the adenoviral vector include,but are not limited to, 293 cells (see, e.g., Graham, et al. (1977) J.Gen. Virol. 36:59-72), PER.C6 cells (see, e.g., WO 1997/00326, U.S. Pat.Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (see, e.g., WO1995/34671 and Brough, et al. (1997) J. Virol. 71:9206-9213). In someinstances, the complementing cell will not complement for all requiredadenoviral gene functions. Helper viruses can be employed to provide thegene functions in trans that are not encoded by the cellular oradenoviral genomes to enable replication of the adenoviral vector.Adenoviral vectors can be constructed, propagated, and/or purified usingthe materials and methods set forth, for example, in U.S. Pat. Nos.5,965,358, 5,994,128, 6,033,908, 6,168,941, 6,329,200, 6,383,795,6,440,728, 6,447,995, 6,475,757, US 2002/0034735, WO 1998/53087, WO1998/56937, WO 1999/15686, WO 1999/54441, WO 2000/12765, WO 2001/77304,and WO 2002/29388, as well as the other references identified herein.Non-group C adenoviral vectors, including adenoviral serotype 35vectors, can be produced using the methods set forth in, for example,U.S. Pat. Nos. 5,837,511, 5,849,561, WO 1997/12986 and WO 1998/53087.Moreover, numerous adenoviral vectors are available commercially.

The adenoviral vector's coat protein can be modified so as to decreasethe adenoviral vector's ability or inability to be recognized by aneutralizing antibody directed against the wild-type coat protein. Suchmodifications are useful for multiple rounds of administration.Similarly, the coat protein of the adenoviral vector can be manipulatedto alter the binding specificity or recognition of the adenoviral vectorfor a viral receptor on a potential host cell. Such manipulations caninclude deletion or substitution of regions of the fiber, penton, hexon,pIIIa, pVI, and/or pIX, insertions of various native or non-nativeligands into portions of the coat protein, and the like. Manipulation ofthe coat protein can broaden the range of cells infected by theadenoviral vector or enable targeting of the adenoviral vector to aspecific cell type. The ability of an adenoviral vector to recognize apotential host cell can be modulated without genetic manipulation of thecoat protein, i.e., through use of a bi-specific molecule. For instance,complexing an adenovirus with a bispecific molecule comprising a pentonbase- or fiber-binding domain and a domain that selectively binds aparticular cell surface binding site enables the targeting of theadenoviral vector to a particular cell type.

Preferably, the adenoviral capsid is modified to display a non-nativeamino acid sequence. The non-native amino acid sequence can be insertedinto or in place of an internal coat protein sequence (e.g., within anexposed loop of an adenoviral fiber protein) or fused to the terminus ofan adenoviral coat protein (e.g., fused to the C-terminus of anadenoviral fiber protein, optionally using a linker or spacer sequence).The non-native amino acid sequence can be conjugated to any of theadenoviral coat proteins to form a chimeric coat protein. Therefore, forexample, the non-native amino acid sequence of the invention can beconjugated to, inserted into, or attached to a fiber protein, a pentonbase protein, a hexon protein, proteins IX, VI, or IIIa, etc. Thesequences of such proteins, and methods for employing them inrecombinant proteins, are well known in the art (see, e.g., U.S. Pat.Nos. 5,543,328, 5,559,099, 5,712,136, 5,731,190, 5,756,086, 5,770,442,5,846,782, 5,962,311, 5,965,541, 5,846,782, 6,057,155, 6,127,525,6,153,435, 6,329,190, 6,455,314, 6,465,253, 6,576,456, US 2001/0047081,US 2003/0099619, WO 1996/07734, WO 1996/26281, WO 1997/20051, WO1998/07877, WO 1998/07865, WO 1998/40509, WO 1998/54346, WO 2000/15823,WO 2001/58940, and WO 2001/92549). The coat protein portion of thechimeric coat protein can be a full-length adenoviral coat protein towhich the ligand domain is appended, or it can be truncated, e.g.,internally or at the C- and/or N-terminus. The coat protein portion neednot, itself, be native to the adenoviral vector.

Where the ligand is attached to the fiber protein, preferably it doesnot disturb the interaction between viral proteins or fiber monomers.Thus, the non-native amino acid sequence preferably is not itself anoligomerization domain, as such can adversely interact with thetrimerization domain of the adenovirus fiber. Preferably the ligand isadded to the virion protein, and is incorporated in such a manner as tobe readily exposed to the substrate (e.g., at the N- or C-terminus ofthe protein, attached to a residue facing the substrate, positioned on apeptide spacer to contact the substrate, etc.) to maximally present thenon-native amino acid sequence to the substrate. Ideally, the non-nativeamino acid sequence is incorporated into an adenoviral fiber protein atthe C-terminus of the fiber protein (and attached via a spacer) orincorporated into an exposed loop (e.g., the HI loop) of the fiber tocreate a chimeric coat protein. Where the non-native amino acid sequenceis attached to or replaces a portion of the penton base, preferably itis within the hypervariable regions to ensure that it contacts thesubstrate. Where the non-native amino acid sequence is attached to thehexon, preferably it is within a hypervariable region (Miksza, et al.(1996) J. Virol. 70(3):1836-44). Use of a spacer sequence to extend thenon-native amino acid sequence away from the surface of the adenoviralparticle can be advantageous in that the non-native amino acid sequencecan be more available for binding to a receptor and any stericinteractions between the non-native amino acid sequence and theadenoviral fiber monomers is reduced.

A chimeric viral coat protein comprising a non-native ligand isdesirably able to direct entry into cells of the viral, i.e.,adenoviral, vector comprising the coat protein that is more efficientthan entry into cells of a vector that is identical except forcomprising a wild-type viral coat protein rather than the chimeric viralcoat protein. Preferably, the chimeric virus coat protein binds a novelendogenous binding site present on the cell surface that is notrecognized, or is poorly recognized by a vector comprising a wild-typecoat protein.

In addition, the adenoviral capsid proteins can be altered to reduce orablate binding to native adenoviral receptors (i.e., receptors bound bywild-type adenovirus). In particular, the portion of the adenoviralfiber protein which interacts with the coxsackie and adenovirus receptor(CAR) can be mutated by deletion, substitution, repositioning within thefiber protein, etc., such that the adenoviral fiber protein does notbind CAR. Likewise, the portion of the adenoviral penton protein thatinteracts with integrins can be altered to ablate native integrinbinding. To reduce native binding and transduction of thereplication-deficient or conditionally-replicating adenoviral vector,the native binding sites located on adenoviral coat proteins whichmediate cell entry, e.g., the fiber and/or penton base, are absent ordisrupted. Two or more of the adenoviral coat proteins are believed tomediate attachment to cell surfaces (e.g., the fiber and penton base).Any suitable technique for altering native binding to a host cell (e.g.,a mesothelial cell or hepatocyte) can be employed. For example,exploiting differing fiber lengths to ablate native binding to cells canbe accomplished via the addition of a binding sequence to the pentonbase or fiber knob. This addition can be done either directly orindirectly via a bispecific or multispecific binding sequence.Alternatively, the adenoviral fiber protein can be modified to reducethe number of amino acids in the fiber shaft, thereby creating a“short-shafted” fiber (as described in, for example, U.S. Pat. No.5,962,311). The fiber proteins of some adenoviral serotypes arenaturally shorter than others, and these fiber proteins can be used inplace of the native fiber protein to reduce native binding of theadenovirus to its native receptor. For example, the native fiber proteinof an adenoviral vector derived from serotype 5 adenovirus can beswitched with the fiber protein from adenovirus serotypes 40 or 41.

In this regard, the adenoviral vector can be modified to include anadenoviral coat protein (e.g., fiber, penton, or hexon protein) from adifferent serotype of adenovirus. For example, an adenoviral serotype 5adenovirus can be modified to display an adenovirus serotype 35 fiber,which, in turn, can optionally comprise one or more non-native aminoacid ligands. It is possible to utilize an adenoviral vector which doesnot naturally infect cell types of the inner ear to target the vector toa particular cell type. Alternatively, an adenoviral vector whichnaturally transduces cells of the inner ear can be modified to displayan adenoviral fiber protein and/or adenoviral penton base derived froman adenovirus which has no natural tropism for target cells, whichadenoviral vector can display a non-native amino acid sequence thatenables transduction of target cells.

In another embodiment, the nucleic acid residues associated with nativesubstrate binding can be mutated (see, e.g., WO 2000/15823; Einfeld, etal. (2001) J. Virol. 75(23):11284-11291; van Beusechem, et al. (2002) J.Virol. 76(6):2753-2762) such that the adenoviral vector incorporatingthe mutated nucleic acid residues is less able to bind its nativesubstrate. For example, adenovirus serotypes 2 and 5 transduce cells viabinding of the adenoviral fiber protein to the coxsackievirus andadenovirus receptor (CAR) and binding of penton proteins to integrinslocated on the cell surface. Accordingly, the replication-deficient orconditionally-replicating adenoviral vector of the inventive method canlack native binding to CAR and/or exhibit reduced native binding tointegrins. To reduce native binding of the replication-deficient orconditionally-replicating adenoviral vector to host cells, the nativeCAR and/or integrin binding sites (e.g., the RGD sequence located in theadenoviral penton base) are removed or disrupted.

Modifications to adenoviral coat proteins can enhance the resultingadenoviral vectors' ability to evade the host immune system. In oneembodiment, the adenoviral vector is selectively targeted to scarredepithelial cells (e.g., regions of the epithelium missing endogenous,functional hair cells) by ablation of native binding of the adenoviralvector to CAR and/or integrins and incorporation into the adenoviralcapsid one or more non-native ligands. Suitable ligands that mediatetransduction via a specific receptor can be determined using routinelibrary display techniques (such as phage display) and include, forexample, ligands bound by EGF and ligands from the FGF family ofpeptides. Other examples of non-native amino acid sequences and theirsubstrates include, but are not limited to, short (e.g., 6 amino acidsor less) linear stretches of amino acids recognized by integrins, aswell as polyamino acid sequences such as polylysine, polyarginine, etc.Non-native amino acid sequences for generating chimeric adenoviral coatproteins are further described in U.S. Pat. No. 6,455,314 and WO2001/92549.

Suitable modifications to an adenoviral vector are described in U.S.Pat. Nos. 5,543,328, 5,559,099, 5,712,136, 5,731,190, 5,756,086,5,770,442, 5,846,782, 5,871,727, 5,885,808, 5,922,315, 5,962,311,5,965,541, 6,057,155, 6,127,525, 6,153,435, 6,329,190, 6,455,314,6,465,253, US 2001/0047081, US 2002/0099024, US 2002/0151027, WO1996/07734, WO 1996/26281, WO 1997/20051, WO 1998/07865, WO 1998/07877,WO 1998/40509, WO 1998/54346, WO 2000/15823, WO 2001/58940, and WO2001/92549. The construction of adenoviral vectors is well understood inthe art. Adenoviral vectors can be constructed and/or purified using themethods set forth, for example, in U.S. Pat. Nos. 5,965,358, 6,168,941,6,329,200, 6,383,795, 6,440,728, 6,447,995, 6,475,757, WO 1998/53087, WO1998/56937, WO 1999/15686, WO 1999/54441, WO 2000/12765, WO 2001/77304,and WO 2002/29388, as well as the other references identified herein.Moreover, numerous expression vectors, including adenoviral vectors, areavailable commercially. Adeno-associated viral vectors can beconstructed and/or purified using the methods set forth, for example, inU.S. Pat. No. 4,797,368 and Laughlin, et al. (1983) Gene 23:65-73.

The selection of an expression vector for use in the inventive methoddepends on a variety of factors such as, for example, the host,immunogenicity of the vector, the desired duration of proteinproduction, the target cell, and the like. As each type of expressionvector has distinct properties, the inventive method can be tailored toany particular situation. Moreover, more than one type of expressionvector can be used to deliver the nucleic acid sequence to the targetcell. Thus, the invention provides method of changing the sensoryperception and preventing or treating hearing loss in an animal, whereinthe method comprises administering to the inner ear at least twodifferent expression vectors comprising a nucleic acid sequence encodingan atonal-associated factor and/or a nucleic acid sequence encoding aninhibitor of EGFR signaling. Preferably, the target cell in the innerear, e.g., a supporting cell, is contacted with an adenoviral vector andan HSV vector, in that adenoviral vectors efficiently transducesupporting cells and HSV vectors efficiently transduce neurons. One ofordinary skill in the art will appreciate the ability to capitalize onthe advantageous properties of multiple delivery systems to treat orstudy sensory disorders of the inner ear.

Nucleic Acid Molecules.

The expression vector of this invention harbors nucleic acid molecules,the expression of which facilitates the regeneration of hair cells andhearing restoration. Ideally, the nucleic acid molecules encode anatonal-associated factor and can further encode an inhibitor of EGFRsignaling. One of ordinary skill in the art will appreciate that anytranscription factor, e.g., Math1 or Hath1 or inhibitor of EGFRsignaling, can be modified or truncated and retain activity. As such,therapeutic fragments (i.e., those fragments having biological activitysufficient to, for example, activate transcription) also are suitablefor incorporation into the expression vector. Likewise, a fusion proteincomposed of a transcription factor or a therapeutic fragment thereofand, for example, a moiety that stabilizes peptide conformation, alsocan be present in the expression vector.

Nucleic acid molecules (i.e., encoding an atonal-associated factorand/or an inhibitor of EGFR signaling) are desirably present as part ofan expression cassette, i.e., a particular base sequence that possessesfunctions which facilitate subcloning and recovery of a nucleic acidmolecule (e.g., one or more restriction sites) or expression of anucleic acid molecule (e.g., polyadenylation or splice sites). When theexpression cassette is an adenoviral vector, the nucleic acid moleculeof interest (e.g., encoding an atonal-associated factor and/or aninhibitor of EGFR signaling) can be located in the E1 region (e.g.,replaces the E1 region in whole or in part) or can be located in the E4region of the adenoviral genome. When positioned in the E4 region, aspacer sequence is not required. The expression cassette is preferablyinserted in a 3′→5′ orientation, e.g., oriented such that the directionof transcription of the expression cassette is opposite that of thesurrounding adenoviral genome. While a single expression cassette can beinserted into an adenoviral vector for expressing an atonal-associatedfactor and/or an inhibitor of EGFR signaling, in other embodiments, theadenoviral vector can include multiple expression cassettes harboringnucleic acid molecules encoding the encoding atonal-associated factorand/or an inhibitor of EGFR signaling, wherein said cassettes canreplace any of the deleted regions of the adenoviral genome. Theinsertion of an expression cassette into the adenoviral genome (e.g.,the E1 region of the genome) can be facilitated by known methods, forexample, by the introduction of a unique restriction site at a givenposition of the adenoviral genome. As set forth above, preferably the E3region of the adenoviral vector is deleted, and the 54 region isreplaced by a spacer element.

For expression, the nucleic acid molecule of interest is operably linkedto regulatory sequences necessary for said expression, e.g., a promoter.A “promoter” is a DNA sequence that directs the binding of RNApolymerase and thereby promotes RNA synthesis. A nucleic acid moleculeis “operably linked” to a promoter when the promoter is capable ofdirecting transcription of that nucleic acid molecule. A promoter can benative or non-native to the nucleic acid molecule to which it isoperably linked. Any promoter (i.e., whether isolated from nature orproduced by recombinant DNA or synthetic techniques) can be used inconnection with the invention to provide for transcription of thenucleic acid molecule. The promoter preferably is capable of directingtranscription in a eukaryotic (desirably mammalian) cell. Thefunctioning of the promoter can be altered by the presence of one ormore enhancers (e.g., the CMV immediate early enhancer) and/orsilencers.

The invention preferentially employs a viral promoter. Suitable viralpromoters are known in the art and include, for instance,cytomegalovirus (CMV) promoters, such as the CMV immediate-earlypromoter, promoters derived from human immunodeficiency virus (HIV),such as the HIV long terminal repeat promoter, Rous sarcoma virus (RSV)promoters, such as the RSV long terminal repeat, mouse mammary tumorvirus (MMTV) promoters, HSV promoters, such as the Lap2 promoter or theherpes thymidine kinase promoter (Wagner, et al. (1981) Proc. Natl.Acad. Sci. USA 78:144-145), promoters derived from SV40 or Epstein Barrvirus, an adeno-associated viral promoter, such as the p5 promoter, andthe like. Preferably, the viral promoter is an adenoviral promoter, suchas the Ad2 or Ad5 major late promoter and tripartite leader, a CMVpromoter (murine or human in origin), or an RSV promoter.

The promoter need not be a viral promoter. For example, the promoter canbe a cellular promoter, i.e., a promoter that drives expression of acellular protein. Preferred cellular promoters for use in the inventionwill depend on the desired expression profile to produce the therapeuticagent(s). In one aspect, the cellular promoter is preferably aconstitutive promoter that works in a variety of cell types. Suitableconstitutive promoters can drive expression of genes encodingtranscription factors, housekeeping genes, or structural genes common toeukaryotic cells. For example, the Ying Yang 1 (YY1) transcriptionfactor (also referred to as NMP-1, NF-E1, and UCRBP) is a ubiquitousnuclear transcription factor that is an intrinsic component of thenuclear matrix (Guo, et al. (1995) Proc. Natl. Acad. Sci. USA92:10526-10530). JEM-1 (also known as HGMW and BLZF-1; Tong, et al.(1998) Leukemia 12(11):1733-1740; Tong, et al. (2000) Genomics69(3):380-390), a ubiquitin promoter, specifically UbC (Marinovic, etal. (2002) J. Biol. Chem. 277(19):16673-16681), a β-actin promoter, suchas that derived from chicken, and the like are appropriate for use inthe inventive method.

Many of the above-described promoters are constitutive promoters.Instead of being a constitutive promoter, the promoter can be aninducible promoter, i.e., a promoter that is up- and/or down-regulatedin response to appropriate signals. For instance, suitable induciblepromoter systems include, but are not limited to, the IL-8 promoter, themetallothionine inducible promoter system, the bacterial lacZYAexpression system, the tetracycline expression system, and the T7polymerase system. Further, promoters that are selectively activated atdifferent developmental stages (e.g., globin genes are differentiallytranscribed from globin-associated promoters in embryos and adults) canbe employed. The promoter sequence that regulates expression of thenucleic acid molecule can contain at least one heterologous regulatorysequence responsive to regulation by an exogenous agent. The regulatorysequences are preferably responsive to exogenous agents such as, but notlimited to, drugs, hormones, or other gene products. For example, theregulatory sequences, e.g., promoter, preferably are responsive toglucocorticoid receptor-hormone complexes, which, in turn, enhance thelevel of transcription of a therapeutic peptide or a therapeuticfragment thereof.

Preferably, the promoter is a tissue-specific promoter, i.e., a promoterthat is preferentially activated in a given tissue and results inexpression of a gene product in the tissue where activated. A tissuespecific promoter for use in this invention can be chosen by theordinarily skilled artisan based upon the target tissue or cell-type.Suitable promoters include, but are not limited to, BRN.3C, BRN 3.1, thePOU ORF3 factor promoter, BRK1, BRK3, the chordin promoter, the nogginpromoter, the jagged1 promoter, the jagged2 promoter, and the notch1promoter. Preferred tissue-specific promoters for use in this inventionare specific to supporting cells or sensory hair cells, such as anatonal promoter or a myosin VIIa promoter, which function in hair cells,or a hes-1 promoter, which functions in supporting cells. Ideally, apromoter is selected that promotes transgene expression in scarredepithelium.

A promoter also can be selected for use in this invention by matchingits particular pattern of activity with the desired pattern and level ofexpression of the desired protein (e.g., the atonal-associated factor).Alternatively, a hybrid promoter can be constructed which combines thedesirable aspects of multiple promoters. For example, a CMV-RSV hybridpromoter combining the CMV promoter's initial rush of activity with theRSV promoter's high maintenance level of activity is especiallypreferred for use in many embodiments of the inventive method. It isalso possible to select a promoter with an expression profile that canbe manipulated by an investigator.

Along these lines, to optimize protein production, preferably thenucleic acid molecule further includes a polyadenylation site followingthe coding region of the nucleic acid molecule. Any suitablepolyadenylation sequence can be used, including a synthetic optimizedsequence, as well as the polyadenylation sequence of BGH (Bovine GrowthHormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein BarrVirus), and the papillomaviruses, including human papillomaviruses andBPV (Bovine Papilloma Virus). A preferred polyadenylation sequence isthe SV40 (Human Sarcoma Virus-40) polyadenylation sequence. Also,preferably all the proper transcription signals (and translationsignals, where appropriate) are correctly arranged such that the nucleicacid molecule is properly expressed in the cells into which it isintroduced. If desired, the nucleic acid molecule also can incorporatesplice sites (i.e., splice acceptor and splice donor sites) tofacilitate mRNA production. Moreover, if the nucleic acid moleculeencodes a protein or peptide, which is a processed or secreted proteinor acts intracellularly, preferably the nucleic acid molecule furtherincludes the appropriate sequences for processing, secretion,intracellular localization, and the like.

In certain embodiments, it may be advantageous to modulate expression ofthe atonal-associated factor and/or inhibitor of EGFR signaling. Anespecially, preferred method of modulating expression of a nucleic acidmolecule involves the addition of site-specific recombination sites onthe expression vector. Contacting an expression vector havingsite-specific recombination sites with a recombinase will either up- ordown-regulate transcription of a coding sequence, or simultaneouslyup-regulate transcription of one coding sequence and down-regulatetranscription of another, through the recombination event. Use ofsite-specific recombination to modulate transcription of a nucleic acidsequence is described in, for example, U.S. Pat. Nos. 5,801,030,6,063,627 and WO 97/09439.

Several options are available for delivering nucleic acid moleculesencoding the atonal-associated factor and/or inhibitor of EGFR signalingto the inner ear. The nucleic acid molecule encoding theatonal-associated factor can also encode an inhibitor of EGFR signaling.The expression vector alternatively, or in addition, can includemultiple expression cassettes encoding atonal-associated factor and/oran inhibitor of EGFR signaling. The multiple coding sequences can beoperably linked to different promoters, e.g., different promoters havingdissimilar levels and patterns of activity. Alternatively, the multiplecoding sequences can be operably linked to the same promoter to form apolycistronic element. The invention also contemplates administering tothe inner ear a cocktail of expression vectors, wherein each expressionvectors encode an atonal-associated factor and/or an inhibitor of EGFRsignaling. The cocktail of expression vectors can further includedifferent types of expression vectors, e.g., adenoviral vectors andadeno-associated viral vectors.

In view of the above, the invention further provides an adenoviralvector harboring a nucleic acid molecule(s) encoding anatonal-associated factor (e.g., Math1 or Hath1) and/or an inhibitor ofEGFR signaling, wherein the nucleic acid molecule(s) is operably linkedto regulatory sequences necessary for expression of theatonal-associated factor and/or inhibitor of EGFR signaling. Theadenoviral vector is deficient in at least one replication-essentialgene function of at least the E4 region. The nucleic acid molecule canbe obtained from any source, e.g., isolated from nature, syntheticallygenerated, isolated from a genetically engineered organism, and thelike. Appropriate adenoviral vectors and regulatory sequences arediscussed herein.

Moreover, the invention further provides a method of generating a haircell in differentiated sensory epithelia in vivo. The method involvescontacting differentiated sensory epithelial cells with an adenoviralvector (a) deficient in one or more replication-essential gene functionsof the E1 region, the E4 region, and, optionally, one or more genefunctions the E3 region, (b) having a spacer in the E4 region, and (c)harboring a nucleic acid molecule(s) encoding an atonal-associatedfactor and/or an inhibitor of EGFR signaling. The nucleic acidmolecule(s) is expressed to produce the atonal-associated factor and/orinhibitor of EGFR signaling such that a hair cell is generated. Whilethe adenoviral vector can be used to generate hair cells in vivo (andtherefore is useful for prophylactically or therapeutically treat ahearing disorder or a balance disorder), transdifferentiation ofsupporting cells can occur in vitro and, thus, can be used in methods ofresearch.

Routes of Administration.

One skilled in the art will appreciate that suitable methods ofadministering a drug or an expression vector, such as an adenoviralvector, to the inner ear are available. Although more than one route canbe used to administer a particular drug or expression vector, aparticular route can provide a more immediate and more effectivereaction than another route. Accordingly, the described routes ofadministration are merely exemplary and are in no way limiting.

No matter the route of administration, a drug or expression vector ofthe inventive method ideally reaches the sensory epithelium of the innerear. The most direct routes of administration, therefore, entailsurgical procedures which allow access to the interior of the structuresof the inner ear. Inoculation via cochleostomy allows administration ofan expression vector directly to the regions of the inner ear associatedwith hearing. Cochleostomy involves drilling a hole through the cochlearwall, e.g., in the otic capsule below the stapedial artery as describedin Kawamoto, et al. ((2001) Molecular Therapy 4(6):575-585), and releaseof a pharmaceutical composition containing the drug or expressionvector. Administration to the endolymphatic compartment is particularlyuseful for administering an adenoviral vector to the areas of the innerear responsible for hearing. Alternatively, a drug or expression vectorcan be administered to the semicircular canals via canalostomy.Canalostomy provides for transgene expression in the vestibular systemand the cochlea, whereas cochleostomy does not provide as efficienttransduction in the vestibular space. The risk of damage to cochlearfunction is reduced using canalostomy in as much as direct injectioninto the cochlear space can result in mechanical damage to hair cells(Kawamoto, et al., supra). Administration procedures also can beperformed under fluid (e.g., artificial perilymph), which can includefactors to alleviate side effects of treatment or the administrationprocedure, such as apoptosis inhibitors or anti-inflammatories.

Another direct route of administration to the inner ear is through theround window, either by injection or topical application to the roundwindow. Administration via the round window is especially preferred fordelivering a drug or adenoviral vector to the perilymphatic space.Transgene expression in cochlear and vestibular neurons and cochlearsensory epithelia has been observed following administration ofexpression vectors via the round window (Staecker, et al. (2001) ActaOtolaryngol. 121:157-163). Of note, it appears possible that uptake ofexpression vectors, in particular non-targeted adenoviral vectors, intocells of the inner ear is not receptor-mediated. In other words, it doesnot appear that adenoviral infection of cells of the inner ear ismediated by CAR or integrins. To increase transduction of cells in theOrgan of Corti following administration to the perilymphaticcompartment, an adenoviral vector can display one or more ligands thatenhance uptake of the adenoviral vector into target cells (e.g.,supporting cells, cells of the stria vascularis, etc.). In this regard,the adenoviral vector can encode one or more adenoviral coat proteinswhich are modified to reduce native binding (e.g., CAR- and/orintegrin-binding) and harbor a non-native amino acid sequence whichenhances uptake of the adenoviral vector by target cells of the innerear.

A drug or expression vector (e.g., adenoviral vector) can be present ina pharmaceutical composition for administration to the inner ear. Incertain cases, it may be appropriate to administer multiple applicationsand/or employ multiple routes, e.g., canalostomy and cochleostomy, toensure sufficient exposure of supporting cells to the drug or expressionvector.

A drug or expression vector can be present in or on a device that allowscontrolled or sustained release of the drug or expression vector, suchas a sponge, meshwork, mechanical reservoir or pump, or mechanicalimplant. For example, a biocompatible sponge or gelform soaked in apharmaceutical composition containing the drug or expression vector isplaced adjacent to the round window, through which the drug orexpression vector permeates to reach the cochlea (as described in Jero,et al., supra). Mini-osmotic pumps provide sustained release of a drugor expression vector over extended periods of time (e.g., five to sevendays), allowing small volumes of composition containing the drug orexpression vector to be administered, which can prevent mechanicaldamage to endogenous sensory cells. The drug or expression vector alsocan be administered in the form of sustained-release formulations (see,e.g., U.S. Pat. No. 5,378,475) containing, for example, gelatin,chondroitin sulfate, a polyphosphoester, such asbis-2-hydroxyethyl-terephthalate (BHET), or a polylactic-glycolic acid.

Alternatively, the drug or expression vector can be administeredparenterally, intramuscularly, intravenously, orally orintraperitoneally. Preferably, a drug or expression vector that isparenterally administered to a patient for generating sensory hair cellsin the ear is specifically targeted to sensory epithelial cells, such assupporting cells. Desirably, the expression vector is targeted toscarred sensory epithelium to promote generation of exogenous hair cellsto replace damaged endogenous hair cells. As discussed herein, anexpression vector can be modified to alter the binding specificity orrecognition of an expression vector for a receptor on a potential hostcell. With respect to adenovirus, such manipulations can includedeletion of regions of the fiber, penton, or hexon, insertions ofvarious native or non-native ligands into portions of the coat protein,and the like. One of ordinary skill in the art will appreciate thatparenteral administration can require large doses or multipleadministrations to effectively deliver the expression vector to theappropriate host cells. Pharmaceutically acceptable carriers forcompositions are well-known to those of ordinary skill in the art (seePharmaceutics and Pharmacy Practice, (1982) J. B. Lippincott Co.,Philadelphia, PA, Banker and Chalmers, eds., pages 238-250; ASHPHandbook on Injectable Drugs (1986) Toissel, 4^(th) ed., pages 622-630).Although less preferred, the expression vector can also be administeredin vivo by particle bombardment, i.e., a gene gun.

One of ordinary skill in the art also will appreciate that dosage androutes of administration can be selected to minimize loss of expressionvector due to a host's immune system. For example, for contacting targetcells in vivo, it can be advantageous to administer to a host a nullexpression vector (i.e., an expression vector not harboring the nucleicacid molecule(s) of interest) prior to performing the inventive method.Prior administration of null expression vectors can serve to create animmunity in the host to the expression vector hinder the body's innateclearance mechanisms, thereby decreasing the amount of vector cleared bythe immune system.

Dosage.

The dose of a drug or expression vector administered to an animal,particularly a human, in accordance with the invention should besufficient to affect the desired response in the animal over areasonable time frame. One skilled in the art will recognize that dosagewill depend upon a variety of factors, including the age, species,location of damaged sensory epithelia, the pathology in question (ifany), and condition or disease state. Dosage also depends on theatonal-associated factor, inhibitor of EGFR signaling and/or cellcycle-associated protein kinase inhibitor, as well as the amount ofsensory epithelium to be transduced. The size of the dose also will bedetermined by the route, timing, and frequency of administration as wellas the existence, nature, and extent of any adverse side effects thatmight accompany the administration of a particular expression vector(e.g., surgical trauma) or drug and the desired physiological effect. Itwill be appreciated by one of ordinary skill in the art that variousconditions or disease states, in particular, chronic conditions ordisease states, may require prolonged treatment involving multipleadministrations.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.When the expression vector is a viral vector, most preferably anadenoviral vector, about 10⁵ viral particles to about 10¹² viralparticles are delivered to the patient. In other words, a pharmaceuticalcomposition can be administered that includes an expression vectorconcentration of about 10⁵ particles/ml to about 10¹³ particles/ml(including all integers within the range of about 10⁵ particles/ml toabout 10¹³ particles/ml), preferably about 10¹⁰ particles/ml to about10¹² particles/ml, and will typically involve the administration ofabout 0.1 μl to about 100 μl of such a pharmaceutical compositiondirectly to the inner ear. In view of the above, the dose of oneadministration preferably is at least about 1×10⁶ particles (e.g., about4×10⁶-4×10¹² particles), more preferably at least about 1×10⁷ particles,more preferably at least about 1×10⁸ particles (e.g., about 4×10⁸-4×10¹¹particles), and most preferably at least about 1×10⁹ particles to atleast about 1×10¹⁰ particles (e.g., about 4×10⁹-4×10¹⁰ particles) of anadenoviral vector harboring a nucleic acid molecule encoding anatonal-associated factor and/or a co-transcription factor and/orinhibitor of a gene silencing complex. Alternatively, the dose of thepharmaceutical composition includes no more than about 1×10¹⁴ particles,preferably no more than about 1×10¹³ particles, even more preferably nomore than about 1×10¹² particles, even more preferably no more thanabout 1×10¹¹ particles, and most preferably no more than about 1×10¹⁰particles (e.g., no more than about 1×10⁹ particles). In other words, asingle dose of pharmaceutical composition can be about 1×10⁶ particleunits (pu), 4×10⁶ pu, 1×10⁷ pu, 4×10⁷ pu, 1×10⁸ pu, 4×10⁸ pu, 1×10⁹ pu,4×10⁹ pu, 1×10¹⁰ pu, 4×10¹⁰ pu, 1×10¹¹ pu, 4×10¹¹ pu, 1×10¹¹ pu, 4×10¹¹pu, 1×10¹² pu, or 4×10¹² pu of the adenoviral vector (e.g., thereplication-deficient adenoviral vector). When the expression vector isa plasmid, preferably about 0.5 ng to about 1000 μg of DNA isadministered. More preferably, about 0.1 μg to about 500 μg isadministered, even more preferably about 1 μg to about 100 μg of DNA isadministered. Most preferably, about 50 μg of DNA is administered to theinner ear. Of course, other routes of administration may require smalleror larger doses to achieve a therapeutic effect. Any necessaryvariations in dosages and routes of administration can be determined bythe ordinarily skilled artisan using routine techniques known in theart.

The interior space of the structures of the inner ear is limited. Thevolume of pharmaceutical composition administered directly into theinner ear structures should be carefully monitored, as forcing too muchcomposition will damage the sensory epithelium. For a human patient, thevolume administered is preferably about 10 μl to about ml (e.g., fromabout 25 μl to about 1.5 ml) of composition. For example, from about 50μl to about 1 ml (e.g., about 100 μl, 200 μl, 300 μl, 400 μl, 500 μl,600 μl, 700 μl, 800 μl or 900 μl) of composition can be administered. Inone embodiment, the entire fluid contents of the inner ear structure,e.g., the cochlea or semi-circular canals, is replaced withpharmaceutical composition. In another embodiment, a pharmaceuticalcomposition of the invention is slowly released into the inner earstructure, such that mechanical trauma is minimized.

It can be advantageous to administer two or more (i.e., multiple) dosesof the drug or expression vector harboring a nucleic acid moleculeencoding an atonal-associated factor and/or an inhibitor of EGFRsignaling. The inventive method provides for administration of multipledoses of a drug or expression vector to generate hair cells in thesensory epithelium to change the sensory perception of an animal. Forexample, at least two doses of a drug or expression vector can beadministered to the same ear. Preferably, the multiple doses areadministered while retaining gene expression above background levels.Also preferably, the sensory epithelium of the inner ear is contactedwith two doses or more of the drug or expression vector within about 30days. More preferably, two or more applications are administered to theinner ear within about 90 days. However, three, four, five, six, or moredoses can be administered in any time frame (e.g., 2, 7, 10, 14, 21, 28,35, 42, 49, 56, 63, 70, 77, 85 or more days between doses).

Pharmaceutical Composition.

A drug or expression vector of the invention desirably is administeredin a pharmaceutical composition, which includes a pharmaceuticallyacceptable carrier and the drug or expression vector(s). Any suitablepharmaceutically acceptable carrier can be used within the context ofthe invention, and such carriers are well known in the art. The choiceof carrier will be determined, in part, by the particular site to whichthe composition is to be administered and the particular method used toadminister the composition. Ideally, in the context of adenoviralvectors, the pharmaceutical composition preferably is free ofreplication-competent adenovirus.

Suitable formulations include aqueous and non-aqueous solutions,isotonic sterile solutions, which can contain anti-oxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood or fluid of the inner ear of the intended recipient, and aqueousand non-aqueous sterile suspensions that can include suspending agents,solubilizers, thickening agents, stabilizers, and preservatives. Theformulation can include artificial endolymph or perilymph, which arecommercially available. The formulations can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials, and can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water, immediatelyprior to use. Extemporaneous solutions and suspensions can be preparedfrom sterile powders, granules, and tablets of the kind previouslydescribed. Preferably, the pharmaceutically acceptable carrier is abuffered saline solution. More preferably, the expression vector for usein the inventive method is administered in a pharmaceutical compositionformulated to protect the expression vector from damage prior toadministration. For example, the pharmaceutical composition can beformulated to reduce loss of the expression vector on devices used toprepare, store, or administer the expression vector, such as glassware,syringes, or needles. The pharmaceutical composition can be formulatedto decrease the light sensitivity and/or temperature sensitivity of theexpression vector. To this end, the pharmaceutical compositionpreferably includes a pharmaceutically acceptable liquid carrier, suchas, for example, those described above, and a stabilizing agent selectedfrom the group consisting of polysorbate 80, L-arginine,polyvinylpyrrolidone, trehalose, and combinations thereof. Use of such apharmaceutical composition will extend the shelf-life of the vector,facilitate administration, and increase the efficiency of the inventivemethod. In this regard, a pharmaceutical composition also can beformulated to enhance transduction efficiency. In addition, one ofordinary skill in the art will appreciate that the expression vector,e.g., viral vector, can be present in a composition with othertherapeutic or biologically-active agents. For example, therapeuticfactors useful in the treatment of a particular indication can bepresent. Factors that control inflammation, such as ibuprofen orsteroids, can be part of the composition to reduce swelling andinflammation associated with in vivo administration of the viral vector.Immune system suppressors can be administered in combination with theinventive method to reduce any immune response to the vector itself orassociated with a disorder of the inner ear. Angiogenic factors,neurotrophic factors, proliferating agents, and the like can be presentin the pharmaceutical composition. Similarly, vitamins and minerals,anti-oxidants, and micronutrients can be co-administered. Antibiotics,i.e., microbicides and fungicides, can be present to reduce the risk ofinfection associated with gene transfer procedures and other disorders.

Other Considerations.

The inventive method includes administering an inhibitor of EGFRsignaling and/or an expression vector(s) harboring a nucleic acidmolecule(s) encoding an atonal-associated factor to change the sensoryperception of an animal by generating hair cells in the sensoryepithelium of the inner ear. The nucleic acid molecule encoding theatonal-associated factor can encode multiple (i.e., two, three, or more)atonal-associated factors and/or inhibitors of EGFR signaling. However,the mere generation of a hair cell does not ensure a change in sensoryperception in an animal. A sufficient number of hair cells should begenerated, and those sensory hair cells should be linked to a neuralnetwork capable of transmitting signals to the brain. Accordingly, whilenot required, it may be advantageous to provide additional factors toensure proper reception and transmission of signals to the brain.

As discussed herein, several options are available for deliveringmultiple coding sequences to the inner ear. The nucleic acid molecule(s)encoding the atonal-associated factor and/or inhibitor of EGFR signalingcan encode additional gene products. The expression vectoralternatively, or in addition, can include multiple expression cassettesencoding different gene products. Multiple coding sequences can beoperably linked to different promoters, e.g., different promoters havingdissimilar levels and patterns of activity. Alternatively, the multiplecoding sequences can be operably linked to the same promoter to form apolycistronic element. The invention also contemplates administering tothe inner ear a cocktail of expression vectors, wherein each expressionvector encodes an atonal-associated factor or another gene productbeneficial to sensory perception. The cocktail of expression vectors canfurther comprise different types of expression vectors, e.g., adenoviralvectors and adeno-associated viral vectors.

Alternatively, or in addition to the administration of an inhibitor ofEGFR signaling and/or an expression vector(s) harboring a nucleic acidmolecule(s) encoding an atonal-associated factor, the inventive methodalso includes the administration of an expression vector harboring anucleic acid molecule encoding an atonal-associated factor incombination with an inhibitor of EGFR signaling, which is not encoded byan expression vector. In this respect, the method and pharmaceuticalcompositions disclosed herein can be modified to include the expressionvector harboring a nucleic acid molecule encoding an atonal-associatedfactor in combination with an isolated inhibitory RNA molecule, protein,peptide, antibody, or small organic molecule that inhibits EGFRsignaling. Moreover, the method and pharmaceutical compositionsdisclosed herein can be modified to include (i) the expression vector(s)harboring a nucleic acid molecule(s) encoding an atonal-associatedfactor and/or an inhibitor of EGFR signaling in combination with (ii) anisolated inhibitory RNA molecule, protein, peptide, antibody, or smallorganic molecule that inhibits EGFR signaling.

For carrying out the methods disclosed herein, this invention alsoprovides a kit. Ideally, the kit includes a nucleic acid moleculeencoding an atonal-associated factor, and (i) a nucleic acid moleculeencoding an inhibitor of epidermal growth factor receptor (EGFR)signaling; (ii) an isolated inhibitor of EGFR signaling (e.g., aninhibitory RNA or small organic molecule that inhibits one or more ofEGFR, Ras, Raf, MEK, ERK/MAPK, JAK, STAT, PI3K, AKT, mTOR, NCK, PAK,JNK, PLC, PKC or a cell cycle associated kinase); or (iii) a combinationof (i) and (ii). In addition, the kit can include containers (e.g.,vials, bottles, syringes, or tubes) containing the active ingredients inlyophilized or liquid form as well as instructions for using the kitcomponents including information regarding dosing, administration,timing of administration and the like. The kit may further include anindication of the active ingredients, reference to scientificliterature, packing materials, clinical trial results, and the like. Theinformation may be based on the results of various studies, for example,studies using experimental animals involving in vivo models and studiesbased on human clinical trials.

In one preferred embodiment, the inventive method also contemplatesdelivery of a nucleic acid molecule encoding at least one neurotrophicagent. Ideally, the neurotrophic agent is a neural growth stimulator,which induces growth, development, and/or maturation of neuralprocesses. Neurotrophic factors also can be administered to protect ormaintain existing and developing neurons. For a newly generated haircell to function properly, a neural network should be in place totransmit neural impulses to the brain. Accordingly, it is advantageousto protect existing neurons associated with the sensory epithelium ofthe inner ear while generating new hair cells, induce the growth andmaturation of new neural processes, and/or simply direct existing neuralprocesses to sensory hair cells. Neurotrophic factors are divided intothree subclasses: neuropoietic cytokines; neurotrophins; and thefibroblast growth factors. Ciliary neurotrophic factor (CNTF) isexemplary of neuropoietic cytokines. CNTF promotes the survival ofciliary ganglionic neurons and supports certain neurons that areNGF-responsive. Neurotrophins include, for example, brain-derivedneurotrophic factor (BDNF) and nerve growth factor (NGF), whichstimulates neurite outgrowth. Other neurotrophic factors include, forexample, transforming growth factors, glial cell-line derivedneurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4/5, andinterleukin 1-β. Neuronotrophic factors enhance neuronal survival andalso are suitable for use in the inventive method. It has beenpostulated that neuronotrophic factors can actually reverse degradationof neurons. Such factors, conceivably, are useful in treating thedegeneration of neurons associated with age, infection, or trauma. Apreferred neuronotrophic factor is pigment epithelium derived factor(PEDF). PEDF is further described in Chader (1987) Cell Different.20:209-216; Pignolo, et al. (1998) J. Biol. Chem. 268(12):8949-8957;U.S. Pat. No. 5,840,686, WO 1993/24529, WO 1999/04806, and WO2001/58494.

Proliferating agents induce cellular proliferation, preferablyproliferation of supporting cells in the inner ear. Multiplying thenumber of hair cell progenitors maximizes the biological effect of theatonal-associated factor and/or a co-transcription factor and/orinhibitor of a gene silencing complex. Supporting cell proliferation isinduced by mitogenic growth factors, such as fibroblast growth factors(FGF, in particular FGF-2), vascular endothelial growth factors (VEGF),epidermal growth factor (EGF), E2F, cell cycle up-regulators, and thelike. A nucleic acid sequence encoding a proliferating agent can beadministered in conjunction with the nucleic acid molecule(s) encodingan atonal-associated factor and/or an inhibitor of EGFR signaling in theinventive method. If desired, the nucleic acid molecule encoding aproliferating agent can be engineered to exert its biological effectonly on the cell type to be replicated. For supporting cells, thenucleic acid can include a regulatory sequence that is preferentiallyactivated in supporting cells. The resulting proliferating agent alsocan be engineered to prevent secretion into the cellular milieu.Alternatively, a substance can be administered to the inner ear topromote cell proliferation or enhance uptake of the expression vector.

The method of the invention can be part of a treatment regimen involvingother therapeutic modalities. It is appropriate, therefore, if theinventive method is employed to prophylactically or therapeuticallytreat a sensory disorder, namely a hearing disorder or a balancedisorder, that has been treated, is being treated, or will be treatedwith any of a number of other therapies, such as drug therapy orsurgery. The inventive method also can be performed in conjunction withthe implantation of hearing devices, such as cochlear implants. Theinventive method also is particularly suited for procedures involvingstem cells to regenerate populations of cells within the inner ear. Inthis respect, the inventive method can be practiced ex vivo to transducestem cells, which are then implanted within the inner ear.

The expression vector is preferably administered as soon as possibleafter it has been determined that an animal, such as a mammal,specifically a human, is at risk for degeneration of sensory hair cells(prophylactic treatment) or has demonstrated reduced numbers or damageof sensory hair cells (therapeutic treatment). Treatment will depend, inpart, upon the particular nucleic acid molecule used, the particularatonal-associated factor and/or inhibitor of EGFR signaling expressed,the expression vector, the route of administration, and the cause andextent, if any, of hair cell loss or damage realized.

An expression vector(s) harboring a nucleic acid molecule(s) encoding anatonal-associated factor and/or an inhibitor of EGFR signaling can beintroduced ex vivo into cells previously removed from a given animal, inparticular a human. Such transduced autologous or homologous host cellscan be progenitor cells that are reintroduced into the inner ear of theanimal or human to express the atonal-associated factor and/or inhibitorof EGFR signaling and differentiate into mature hair cells in vivo. Oneof ordinary skill in the art will understand that such cells need not beisolated from the patient, but can instead be isolated from anotherindividual and implanted into the patient.

The inventive method also can involve the co-administration of otherpharmaceutically active compounds. By “co-administration” is meantadministration before, concurrently with, e.g., in combination with theexpression vector in the same formulation or in separate formulations,or after administration of the expression vector as described above. Forexample, factors that control inflammation, such as ibuprofen orsteroids, can be co-administered to reduce swelling and inflammationassociated with administration of the expression vector.Immunosuppressive agents can be co-administered to reduce inappropriateimmune responses related to an inner ear disorder or the practice of theinventive method. Similarly, vitamins and minerals, anti-oxidants, andmicronutrients can be co-administered. Antibiotics, i.e., microbicidesand fungicides, can be co-administered to reduce the risk of infectionassociated with surgical procedures.

The following non-limiting examples are provided to further illustratethe present invention.

Example 1: Small Molecule Inhibition of EGFR Signaling for Hair CellRegeneration

Atoh1, a transcription factor, can convert non-sensory supporting cells(SCs) to hair cells (HCs) in neonatal and juvenile mammalian cochleae(Izumikawa, et al. (2005) Nature Med. 11:271-6; Kelly, et al. (2012) J.Neurosci. 32:6699-710; Liu, et al. (2012) J. Neurosci. 32:6600-10).Ectopic expression of Atoh1 has been approved for clinical trials as agene therapy. However, Atoh1-induced HC conversion is inefficient(<17%), incomplete (lacking mature HC markers), and age-dependent (noresponse in adult cochlea) (Izumikawa, et al. (2008) Hearing Res.240:52-6; Liu, et al. (2012) J. Neurosci. 32:6600-10). Using RNA-seqanalysis to compare the transcriptome of SCs, endogenous outer HCs(OHCS), and Atoh1-induced HCs (cHCs) in adult mouse cochleae, it wasobserved that the epidermal growth factor receptor (EGFR) signaling isenriched in SCs and cHCs using gene set enrichment analysis (GSEA). Tovalidate the role of EGFR pathway ex vivo, neonatal mouse cochlearexplants were treated with a potent EGFR inhibitor (AG1478). Thisanalysis indicated that, by itself, AG1478 had no impact on theproliferation and differentiation of endogenous SCs and HCs after 7days. However, when AG1478 was combined Atoh1, there was a significantincrease in Atoh1-induced HC conversion from explant SCs (from 24.7% to80.6%; FIGS. 1A-1D). In addition, inhibitors targeting proteinsdownstream of EGFR signaling were tested including WP1066 (STAT3inhibitor), LY 294002 (PI3K inhibitor), U0126 (MEK inhibitor), andU73122 (PLC inhibitor) at various doses above their respective EC₅₀values. With the exception of PLC inhibition, a similar enhancement ofconversion rate of Atoh1-induced HCs was observed (FIG. 2 ). Thisanalysis indicates that EGFR signaling pathways play novel roles in SCconversion to HCs. While known to be important in development(Doetzlhofer, et al. (2004) Dev. Biol. 272:432-47; Yamashita & Oesterle(1995) Proc. Natl. Acad. Sci. USA 92:3152-5), EGFR signaling has notpreviously been implicated in HC regeneration.

To further evaluate the use of an inhibitor of EGFR signaling in HCregeneration, the toxicity of AG1478 in neonatal and adult mice wasassessed. Based upon this analysis, the median lethal dose (LD₅₀) ofAG1478 for postnatal day 1 (P1) in FVB mice was estimated as 30 mg/kgvia intraperitoneal (IP) injection. For adult (>P21) FVB mice, 7 daysafter transtympanic (TT) injection, 10 μg of AG1478 resulted in nosignificant changes in the auditory brainstem response (ABR) thresholds.

Example 2: Models of EGFR Deficiency in Atoh1-Overexpressing Mice

Prox1-CreER (Prox1), CAG-loxp-stop-loxp-Atoh1-HA (HA), andEGFR^(flox/flox) were crossed to generate an inducible mouse line(Prox1; HA; EGFR^(flox/flox) or PHER) in which Atoh1 overexpressiontogether with EGFR deletion can be achieved specifically in neonatal andjuvenile SCs (Deiters cells (DC) and pillar cells (PC) underneath outerHCs (OHCS)) upon tamoxifen induction. In 3 and 6 weeks post-induction,the expression of HC markers (early and late) and morphology are assayedby immunohistochemistry, and the electrophysiological profiles of thenewly generated HCs are examined with HA staining.

Similarly, Glast-CreER (Glast) has been used with HA andEGFR^(flox/flox) to generate a mouse line (Glast; HA; EGFR^(flox/flox)or GHER) that specifically targets inner phalangeal and inner bordercells underneath inner HCs (IHCs). In addition, Fgfr3-CreER; HA;EGFR^(flox/flox) or FHER mice were generated to target adult deiter andpillar cells. Cre activity will be induced at P28 adult age and HCregeneration 3 and 6 weeks later will be investigated. It is expectedthat an increase in SC conversion to HC will be observed in micedeficient in EGFR expression.

In addition, one-month-old mice will be exposed to 8-16 kHz octave bandnoise at 120-dB SPL for 2 hours to induce OHC loss and hearing loss.Subsequently, HC regeneration will be induced with tamoxifen 7 dayspost-exposure. The ABR of the animals will be tested before noisedamage, 7 days after noise damage, and 3 weeks after tamoxifen inductionto further determine molecular pathways in HC regeneration in the mousemodels.

Example 3: Therapeutic Potential of EGFR Inhibitors inAtoh1-Overexpressing Mice

Selected EGFR inhibitors will be tested in mouse models with Atoh1overexpression (PH, GH, and FH) to ascertain their potential to mimicthe genetic models described above in Example 2 (i.e., PHER, GHER, andFHER respectively). Six highly potent and specific EGFR inhibitors willbe individually tested including Erlotinib, Gefitinib, Afatinib, NT-113,AZD3759, and Dacomitinib, with EC₅₀ values of 0.2-33 nM. These compoundshave been approved by the FDA or are in clinical trials for otherdiseases, and have relatively well-characterized absorption,distribution, metabolism, excretion, and toxicity (ADMET) propertiesacross species. For this analysis, Atoh1 overexpression will be inducedin SCs with tamoxifen in the mouse models (PH, GH, and FH).Subsequently, the selected compounds will be delivered by IP or TTinjection. ADMET properties of each compound in plasma, cochlearhomogenate, or perilymph will be assessed, and exposure-efficacyrelationships in regenerating functional HCs and restoring hearing afterNIHL will be examined.

It is expected that the use of EGFR inhibitors together withoverexpression of Atoh1 (EGFRi/Atoh1) will generate much more HCs thanAtoh1 alone in neonatal and adult cochleae. It is further expected thata portion of these HCs will express mature HC markers (prestin,oncomodulin, vGlut3) and exhibit similar electrophysiology as normalmature HCs. EGFRi/Atoh1-mediated functional HC conversion occurs even innoise-induced hearing loss. If the efficiency of EGFR knockout and/orEGFR inhibitors were less than optimal in Atoh1-overexpressing adultSCs, an Atoh1/Pou4f3 overexpressing mouse model can be used, which givesrise to ˜150 cHCs (highest among many models) per cochlea from SCs wheninduced at adult ages. It is expected that the EGFR inhibition willsynergistically promote SC-to-HC conversion with overexpression of bothPou4f3 and Atoh1.

Example 4: EGFR Inhibitors for Protection Against Hearing Loss

A screen of a library composed of 75 kinase inhibitors was conducted toidentify inhibitors that protect against cisplatin-induced hair cellloss. This screen identified four compounds: (1) Her2 inhibitorMUBRITINIB (TAK 165), (2) Pan-AUR inhibitor SNS314 (3) BRAF-V600Einhibitor GSK2118436A (DABRAFENIB), and (4) PDGFR inhibitor CRENOLANIBthat potently protected against cisplatin-induced cell death in a mousecochlea-derived cell line (HEI-OC1) as well as cisplatin-induced haircell loss in cochlear explants. Her2 inhibitor MUBRITINIB (TAK 165)exhibited protective effects against cisplatin-induced Caspase-3/7activity in HEI-OC1 cells with an IC₅₀ of 4 nM and LD₅₀ of >55 μM; andprotected against cisplatin-induced hair cell loss in mouse cochlearexplants with IC₅₀ of 2.5 nM and LD₅₀ of >500 nM (Therapeutic Indexof >200)(FIG. 3 ). Similarly, the pan-ErbB inhibitor, PELITINIB, wasfound to exhibit protective effects against cisplatin-inducedCaspase-3/7 activity in HEI-OC1 cell loss with IC₅₀ of 0.6 μM and LD₅₀of 40 μM (FIG. 4 ). Moreover, with 1 hour pre-incubation, PELITINIBexhibited 49% protection of outer hair cells against cisplatin-inducedhair cell loss in mouse cochlear explants (N=3).

Example 5: Protection Against Noise- and Blast Injury-Induced HearingLoss In Vivo in Adult Mouse Models

The protective effects of the top inhibitors of EGFR signaling,administered locally (transtympanic injection into the middle ear), istested against NIHL and blast injury-induced hearing loss in adult mousemodels. The local delivery route is used because it is frequently usedin mammalian hearing studies, as it offers minimal invasiveness andsimple procedures. Specifically, drugs are commonly administered viathis route by pediatricians and ENT doctors to patients of diverse ages(Banerjee & Parnes (2005) Otol. Neurotol. 26:878-881; Dodson, et al.(2004) Ear Nose Throat J. 83:394-398; McCall, et al. (2010) Ear Hear.31:156-165; Muller & Barr-Gillespie (2015) Nat. Rev. Drug Discov.14:346-365; Rauch (2004) Otolaryngol. Clin. North Am. 37:1061-1074).Compounds demonstrating efficacy in murine models can be directly testedfor prevention of cisplatin-associated hearing loss in patientsundergoing cisplatin chemotherapy in clinical trials. Further,transtympanic delivery allows the compounds to diffuse easily across theround window membrane into the endolymphatic fluid (Borkholder (2008)Curr. Opin. Otolaryngol. Head Neck Surg. 16:472-477; Mizutari, et al.(2013) Neuron 77:58-69; Swan, et al. (2008) Adv. Drug Deliv. Rev.60:1583-1599; Tamura, et al. (2005) Laryngoscope 115:2000-2005), suchthat their potency and toxicity can be directly tested in vivo withlittle concern about the blood-labyrinth barrier (BLB). Oral and otherroutes may also be considered for in vivo properties (e.g., solubility,permeability, pharmacokinetics/pharmacodynamics (PK/PD), and absorption,distribution, metabolism, excretion, and toxicology (ADMET)).

Compounds to test can be selected on the basis of: (1) exhibiting potentIC₅₀ values and minimal toxicity (i.e., high LD₅₀/IC₅₀ values,preferably >50-100 μM); (2) targeting several different biologicaltargets/pathways; and (3) capacity to be delivered via other routes(e.g., oral).

For tests of noise injury, wild-type FVB mice are used at age P28, whenhearing has matured but long before significant age-related hearing loss(Kermany, et al. (2006) Hear Res. 220:76-86; Maison, et al. (2002) J.Neurosci. 22:10838-10846; Maison, et al. (2007) J. Neurophysiol.97:2930-2936; Zheng, et al. (1999) Hearing Research 130:94-107). Thestandard noise exposure protocols (94, 100, 106, 116, and 120 dB soundpressure level (SPL) octave-band 8-16 kHz noise for 2 hours) havepreviously been tested in various transgenic mouse strains from the FVBbackground (Maison, et al. (2002) J. Neurosci. 22: 10838-10846; Maison,et al. (2007) J. Neurophysiol. 97:2930-2936). These noise injuryprotocols led to hearing loss (ABR) in CBA/CaJ mice (Wang, et al. (2002)J. Assoc. Res. Otolaryngol. 3:248-268).

Repeated impulses at 135-155 dB SPL can effectively recapitulate theeffects of blast injury in adult mouse cochleae. Previous studies ofblast injuries in animal models have demonstrated that 50-160 repeatedimpulses at 147-160 dB SPL impose physiological and morphological damageto the cochleae of chinchilla, sheep and pigs similar to 3-4 impulses of14 psi blasts (194 peak dB SPL) in rats (Choi, et al. (2008) Free Radic.Biol. Med. 44:1772-1784; Hamernik, et al. (1987) J. Acoust. Soc. Am.81:1118-1129; Henselman, et al. (1994) Hear. Res. 78:1-10; Kopke, et al.(2005) Acta Otolaryngol. 125:235-243; Roberto, et al. (1989) Ann. Otol.Rhinol. Laryngol. Suppl. 140:23-34). More interestingly, 3-4 impulses of14 psi blasts cause 43% OHC loss and 30-40 dB ABR threshold elevation inrats 21 days post-blast, damage that resembles that caused by 6 hours ofcontinuous exposure to 105 dB SPL octave-band noise centered at 4 kHz inchinchillas (Choi, et al. (2008) Free Radic. Biol. Med. 44:1772-1784;Ewert, et al. (2012) Hear. Res. 285:29-39; Kopke, et al. (2005) ActaOtolaryngol. 125:235-243). Based on these results, a range of 135-155 dBSPL octave-band 8-16 kHz noise impulses of ˜10-ms duration are used,which can be repeated 100 times at 1-s intervals in mouse models tomimic traumatic blast injury (Choi, et al. (2008) Free Radic. Biol. Med.44:1772-1784; Ewert, et al. (2012) Hear. Res. 285:29-39; Henselman, etal. (1994) Hear. Res. 78:1-10; McFadden, et al. (2000) J. Acoust. Soc.Am. 107:2162-2168).

Experimental Design.

Mice exposed to noise or blast exposure are immediately treated with theindividual compounds in one ear and vehicle control (0.5% DMSO) in theother ear. DMSO or compound are delivered locally at the highestfeasible dose (which should be much higher than the IC₅₀ in cochlearexplants but not toxic by itself in vivo) by trans-tympanic injectioninto the adult mouse middle ear at ˜5 μL per ear. The ABR and DistortionProducts Otoacoustic Emissions (DPOAE) are measured pre-noise exposureor TBI and at 1 and 2 weeks post-injection. After hearing tests by ABRand DPOAE, the mice are cardiac-perfused for fixation and harvesting ofthe cochleae. The cochleae are analyzed using both whole-mountpreparations and sections, and immunofluorescence is used to detectHC/SC markers (i.e., phalloidin, Myo7a, Prestin, and Sox2, etc.) andsynaptic markers (Ctbp2, GluR2/3 and Tuj1) (Liu, et al. (2014) PLoS One9:e89377).

The entire procedure is double-blinded: one person encodes DMSO orcompound while the other person 1 randomly injects the left and rightears of the same mouse, and the person who records ABR and DPOAE doesnot know which ear was injected with compound until the entireexperiment is completed.

ABR/DPOAE Measurements in Adult Mice. ABR measurements have beenpreviously described in detail (Dallos, et al. (2008) Neuron 58:333-339;Gao, et al. (2007) Mol. Cell Biol. 27:4500-4512; Liberman, et al. (2002)Nature 419:300-304; Liu, et al. (2014) PLoS One 9:e89377; Wu, et al.(2004) Brain Res. Mol. Brain Res. 126:30-37; Yamashita, et al. (2012)PLoS One 7:e45453). Briefly, mice are anesthetized by intraperitonealinjection of AVERTIN (0.5 mg/kg body weight) and placed on an electricheating pad to maintain body temperature, using a homeothermic blanketsystem (Harvard Apparatus Ltd). Mice that die or show signs ofmiddle-ear dysfunction during the course of the experiment are excludedfrom analysis. All recordings are conducted in a sound booth (IndustrialAcoustic Company). For acoustic stimulation and measurements, twospeakers (f1 and f2; EC1) and a microphone (ER-10B, Etymotic Research,Elk Grove Village, IL) are connected to a short flexible coupler tubewith a tapered plastic tip that is inserted into the external auditorymeatus. The microphone is calibrated in situ with the coupler in themeasuring position. At frequencies higher than 22 kHz, the frequencyresponses of the measurement microphone (ER10B+) are lower than those ofa reference microphone (ACO-7017; ACO Pacific, Inc., Belmont, CA).Therefore, DPOAE 2f1-f2 responses are recorded at a frequency f1 rangeof 5454-18180 Hz, using the TDT BioSig III system (TDT). Signal durationis 83.88 ms, with a repetition rate of 11.92/s. The f1 and f2 responsesare passed separately through an RX6 MultiFunction Processor (TDT) fordigital/analog conversion to PA5 programmable attenuators. Stimulusintensity is reduced from 90 to 0 dB in 5 dB steps to establishthresholds and is digitally sampled at 200 kHz and averaged from 100discrete spectra. The signals are delivered through ED1 speaker driversthat feed into the EC1 electrostatic speakers coupled to the ear canal.The resulting ear canal sound pressure is recorded with an ER10B+ lownoise microphone (gain Ox) and probe (Etymotic) housed in the samecoupler as the f1 and f2 speakers. The output of the ER10B+ amplifier isrouted directly to an RX6 MultiFunction Processor (TDT) foranalog/digital conversion for sampling at 200 kHz. Fast-Fouriertransforms (FFT) of averaged responses are generated by using TDTBioSigRP software on the resultant waveform (TDT). Noise floors aredetermined by averaging the sound levels of 10 frequency bins above andbelow the 2f1-f2 frequency bin. No instrumental distortion products havebeen observed in evaluation of ears postmortem.

Noise Injury in Adult Mice.

Mice are placed individually in a cage within a custom-made acrylicchamber in which no two sides are parallel. The sound stimulus isproduced by an RZ6 processor (Tucker-Davis Technologies, GainesvilleFL), filtered (Frequency Devices, Inc., Haverhill, MA), amplified (CrownXTi 1000 amplifier; Crown, Elkhart, IN), and delivered to the acrylicchamber via a speaker horn (JBL, Northridge, CA). The sound pressurelevel is measured through a ¼-inch free-field microphone (ACO Pacific,Belmont, CA) and calibrated to a 124 dB pistonphone (Bruel and Kjaer,Denmark). Prior to experimental noise exposure, four quadrants of thechamber are sampled with the ¼-inch microphone to ensure that soundpressure varies by <0.5 dB across the measured positions.

Trans-Tympanic Injection of Adult Mice.

Mice are anesthetized by intraperitoneal (i.p.) injection of AVERTIN orketamine and xylazine. Body temperature is maintained on a heating padduring the surgical procedure. Lubricant eye ointment is applied toprevent corneal ulcers, as the blinking reflex disappears duringsurgery. The tympanic membrane is visualized with a surgicalstereomicroscope. Using a 33-gauge cannula, 5 μL of compound or DMSO inPBS is gently injected through the tympanic membrane, followed bysurgical stereomicroscopic confirmation that the solution is in themiddle ear cavity. Mice are then placed in the cage on the heating padfor an additional 30 minutes. After surgery, all mice are allowed torecover on a heating pad before being returned to the animal housingfacility.

What is claimed is:
 1. A method for treating cisplatin-induced hearingloss comprising administering to an animal, in conjunction withcisplatin, an inhibitor of epidermal growth factor receptor (EGFR)signaling, wherein the hearing loss is associated with ototoxicity,wherein the inhibitor of EGFR signaling is selected from the groupconsisting of mubritinib, SNS314, dabrafenib and crenolanib.
 2. Themethod of claim 1, further comprising administering one or moreotoprotective or regenerative agents.
 3. A transtympanic pharmaceuticalcomposition in unit dosage form comprising an inhibitor of epidermalgrowth factor receptor (EGFR) signaling, wherein the inhibitor of EGFRsignaling is selected from the group consisting of mubritinib, SNS314,dabrafenib and crenolanib, characterized in that each unit of thepharmaceutical composition is between 100 μl and 900 μl.
 4. Apharmaceutical composition comprising an inhibitor of epidermal growthfactor receptor (EGFR) signaling and one or more regenerative agents,wherein the inhibitor of EGFR signaling is selected from the groupconsisting of mubritinib, SNS314, dabrafenib and crenolanib.
 5. A methodfor treating cisplatin-induced hair cell loss in an ear comprisingadministering to a mammal an inhibitor of EGFR signaling, wherein theinhibitor of EGFR signaling is selected from the group consisting ofmubritinib, SNS314, dabrafenib and crenolanib.
 6. A method for reducingthe risk of cisplatin-induced hearing loss comprising administering toan animal, in conjunction with cisplatin, an inhibitor of epidermalgrowth factor receptor (EGFR) signaling, wherein the hearing loss isassociated with ototoxicity, wherein the inhibitor of EGFR signaling isselected from the group consisting of mubritinib, SNS314, dabrafenib andcrenolanib.
 7. The method of claim 6, further comprising administeringone or more otoprotective or regenerative agents.
 8. A method forreducing the risk of cisplatin-induced hair cell loss in an earcomprising administering to a mammal an inhibitor of EGFR signaling,wherein the inhibitor of EGFR signaling is selected from the groupconsisting of mubritinib, SNS314, dabrafenib and crenolanib.